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Ki-4.dgA is an anti-CD30 immunotoxin (IT) constructed by coupling the monoclonal antibody Ki-4 via a sterically hindered disulfide linker to deglycosylated ricin A-chain. This IT was efficacious in vitro and in SCID mice with disseminated human Hodgkin's lymphoma. Accordingly, a Phase I trial in patients (pts) with Hodgkin's lymphoma was designed. The objectives of this Phase I trial were to determine the maximum tolerated dose, the dose-limiting toxicities, pharmacokinetics, and antitumor activity. Seventeen pts with relapsed CD30+ lymphoma were treated with escalating doses (5, 7.5, or 10 mg/m(2)/cycle) of the IT as four bolus infusions on days 1, 3, 5, and 7 for one to three cycles. All of the pts had progressive disease and were heavily pretreated. Nine had primary progressive disease and 14 had advanced disease with massive tumor burdens. The mean age was 35 years (24-52 years). Peak serum concentrations of the intact IT varied from 0.23 to 1.1 microg/ml. Side effects and dose-limiting toxicities were related to vascular leak syndrome, i.e., decreases in serum albumin, edema, weight gain, hypotension, tachycardia, myalgia, and weakness. The maximum tolerated dose was 5 mg/m(2). Seven of 17 (40%) pts made human antiricin antibodies (> or =1.0 microg/ml), and 1 pt developed human antimouse antibodies (> or =1.0 microg/ml). Clinical response in the 15 evaluable pts included 1 partial remission, 1 minor response, and 2 stable diseases. In conclusion, the IT was less well tolerated than other ITs of this type. This might be because of the low number of CD30+ peripheral blood mononuclear cells, and in part because of binding of the IT to soluble CD30 antigen and the resulting circulation of IT/sCD30 complexes.

New therapeutic approaches for the treatment of B-cell chronic lymphocytic leukemia (B-CLL) can induce remarkable responses. Molecular remissions have been observed occasionally after high-dose chemotherapy. Thus, new improved techniques to monitor residual tumor cells on a molecular basis in CLL are warranted. For this purpose, a real-time quantitative allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) for patients with B-CLL was designed. In the present study, the PCR assay was standardized with identical cycling parameters as well as primer, probe, and MgCl(2) concentration for each patient. Ten patients were monitored with DNA samples obtained at 52 time points (median: 5.2 per patient). The median follow-up per patient was 11.4 months. Nine of ten patients had PCR-detectable residual tumor cells in the peripheral blood after therapy. One patient became PCR negative with a combination of fludarabine and rituximab after the end of treatment. The MRD levels in patients with detectable disease ranged from 0.002% to 10.1% after therapy. We conclude that real-time quantitative ASO-PCR can be utilized for quantitative molecular monitoring of minimal residual disease (MRD) in B-CLL patients in complete remission (CR), that new effective treatment approaches such as combined chemo/immunotherapy can render CLL patients PCR negative, and that different MRD levels in PCR-positive patients were observed warranting further investigation into possible correlation with clinical outcome.

Many new approaches involving antibody-based agents have given promising results in experimental Hodgkin's disease (HD) models. Clinical trials with monoclonal antibodies, immunotoxins, bispecific constructs and radioimmunoconjugates have demonstrated some clinical efficacy in patients with advanced refractory HD. Although it seems unlikely that resistant patients with larger tumor masses will be cured by either of these approaches, it might be feasible to treat bulky disease by conventional therapy and then administer biological agents to kill residual Hodgkin and Reed-Sternberg cells. Future phase III trials will have to prove a possible superior effect of this combined immunochemotherapy. Currently, the evaluation of the most promising approaches continues.

Background: BBR 2778 is a novel aza-anthracenedione showing no cardiotoxicity and superior activity compared to doxorubicin and mitoxantrone in animal models. Objectives of this phase I study included the determination of the maximum tolerated dose (MTD), the dose limiting toxicity (DLT), clinical pharmacokinetics (PK), and antitumor activity. Patients with relapsed or refractory, advanced non-Hodgkin's lymphoma were included., Patients and Methods: Patients were treated with a q1w x 3 schedule on the basis of a modified Fibonacci dose escalation method. Seven groups with a total of twenty-six patients were treated at dosages of 5, 10, 16.5, 25, 34, 56 or 84 mg/m2/w, respectively., Results: DLT was observed on the seventh dose level with neutropenia WHO grade 4 in three of six patients. Pharmacokinetic analysis showed a large volume of distribution (13.5-17.5 l/kg), a high plasma clearance (0.65-1.74 l/h/kg) and a long elimination half-life (14.7-31.9 h). Tumor response included three complete remissions and two partial remissions., Conclusions: Neutropenia is the DLT of the new aza-anthracenedione BBR 2778. The recommend dose is 84 mg/m2 in a q1w x 3 schedule. PK data are consistent with a linear kinetic of BBR 2778 comparable to mitoxantrone. This new drug shows promising activity in intensively pretreated patients with relapsed or refractory NHL. Based on this results, phase II studies with this new compound are underway.

We describe the first clinical application of T-cell-recruiting bispecific antibodies directly into the tumor without the need to preactivate the effector cells. In a Phase I clinical trial, 10 patients with low-grade B-cell lymphoma were treated by a single locoregional injection of CD3xCD19 bispecific antibodies. Costimulatory signaling, which is required for the optimal activation of resting T cells, was provided by the simultaneous administration of CD28 antibodies. Equal amounts of both antibodies were injected together at 4 different dose levels (30 microg: 3 patients; 270 microg: 3 patients; 810 microg: 3 patients; 1,600 microg: 1 patient). The injection was well tolerated with mild to moderate adverse effects (2/10 patients) consisting of erythema and fever at the third dose level. The maximum tolerated dose was not reached at 810 microg of injected antibodies. Three patients showed a serum peak of TNFalpha on day 2 or 3 after the antibody application, reflecting rather an activation of CD4-positive T cells than an FcR-mediated effect. Five patients developed anti-mouse antibodies after injection of the murine immunoglobulins. Nine patients were evaluable for restaging examinations 6 weeks after the antibody application, with 2 of them (22%) showing a local clinical response. We found that a single locoregional injection of CD3xCD19+CD28 antibodies is feasible up to a dose of at least 1,600 microg of each antibody. However, the development of human anti-mouse antibodies points toward the requirement for new formats of bispecific proteins with reduced immunogenicity., (Copyright 2001 Wiley-Liss, Inc.)

The anti-CD25 immunotoxin RFT5.dgA was constructed by coupling the monoclonal antibody RFT5 via a sterically hindered disulfide linker to deglycosylated ricin A-chain and was administered to patients with relapsed Hodgkin's lymphoma in four bolus infusions over 7 days (day 1, 3, 5 and 7). The maximum tolerated dose in these patients as defined in a previous phase I study was 15 mg/m2. Subsequently, further patients were enrolled at the maximum tolerated dose and a total of 18 patients were treated at this level. All patients had signs of progressive disease and were heavily pretreated. Side-effects in this trial were moderate and related to vascular leak syndrome. Five of 18 patients experienced NCI grade III toxicities including weakness, edema, dyspnea, and myalgia. Eleven of 16 (69%) patients receiving two or more cycles produced human anti-ricin antibodies and human anti-mouse antibodies (>/=1.0 microg/ml). Seventeen of 18 patients were evaluable for clinical response. These included two partial remissions. One patient demonstrated minor response and five patients stable diseases. We conclude that RFT5.dgA is of moderate clinical efficacy in this group of heavily pretreated refractory patients. Leukemia (2000) 14, 129-135.

We report a patient with an epidermotropic cutaneous T-cell lymphoma which transformed into an anaplastic cutaneous CD30+ T-cell lymphoma. Repeated relapses required prolonged systemic PUVA therapy. Two years after diagnosis, the patient had several episodes of infections of the respiratory tract. Serum electrophoresis now revealed significantly reduced polyclonal immunglobulin production and an additional band in the gamma fraction corresponding to IgG lambda monoclonal gammopathy. Thereafter, the patient suffered a pathologic fracture of the dorsolateral 5th rib on the right side and an accumulation of monoclonal plasma cells in the bone marrow confirmed the diagnosis of multiple myeloma (IgG lambda). Accordingly, 6 cycles of cytoreductive chemotherapy (alkeran, decortin) were given. After one year of steady state disease the patient lost weight and bone pain increased while only a few papular eruptions were detectable. Radiography showed multiple small osteolytic areas. A few months later he died with signs of bone marrow insufficiency.

We performed a phase II study of dexamethasone, ifosfamide, idarubicin and etoposide (DIZE) in patients with relapsed or refractory Hodgkin's (HL) and non-Hodgkin's lymphoma (NHL). The regimen consisted of dexamethasone (20 mg i.v. days 1-4), idarubicin (8 mg/m2 i.v. days 1+2), continuous infusion (c.i.) of ifosfamide (1,000 mg/m2 days 1-4), and c.i. etoposide (60 mg/m2 days 1-4). G-CSF (5 microg/kg) was used to support neutrophil recovery from day 5. In older patients (> 60 years) the dosage of idarubicin and ifosfamide was reduced to 75% in the initial cycle. Fourty six patients (pts) were treated with a total of 131 cycles. Sixteen pts were primary resistant and 30 were relapsed. Median age was 54.3 years (range 22-75). The median number of different prior chemotherapies was 1.7 (range 1 to 5). 31/46 (67.4%) pts had advanced disease (stage III or IV); 19/46 had B symptoms. Of 43 evaluable pts the response rate was 58.1% including 11 complete remissions (CR) and 14 partial remissions (PR). Mean duration of response was 8 months (1-30+). DIZE was more effective in relapsed than in refractory high-grade NHL (74 % vs 16.6%; p < 0.001). Of four heavily pretreated pts with HL, one obtained CR and two PR (response rate 75%). Myelosuppression was generally moderate with a mean duration of leukocytopenia < 1,000/microl of 2.5 days (range 0-18) and of thrombocytopenia < 25,000/microl 1.5 days (range 0-17). One patient died of uncontrollable infection in treatment related neutropenia. No other serious toxicities apart from alopecia were observed. We conclude that DIZE is safe and effective in heavily pretreated pts with relapsed lymphoma. The continuous infusion of cytostatic drugs such as that used in the new DIZE protocol might reduce hematotoxicity.

Immunotoxins (ITs) consisting of a cell-binding component and a potent toxin were developed as a new class of biological anti-tumor agents to improve adjuvant therapy. Hodgkin's lymphoma (HL) has been demonstrated to be an excellent target for ITs because high concentrations of lymphocyte activation markers such as CD25 and CD30 are expressed on Hodgkin and Reed-Sternberg (H-RS). Several ITs against these antigens have shown potent antitumor effects against H-RS cells in vitro and in different HL animal models. On the basis of its superiority in preclinical models, the anti-CD25 IT RFT5-SMPT-dgA was subsequently evaluated in a phase I study in patients with refractory Hodgkin's lymphoma. The IT was constructed by linking the monoclonal antibody (Moab) RFT5 via a sterically hindered disulfide linker (SMPT) to deglycosylated ricin A-chain (dgA). All 15 patients enrolled in this trial were heavily pretreated with a mean of five different prior therapies. The IT was administered intravenously over four hours on days 1-3-5-7 for total doses per cycle of 5, 10, 15, or 20 mg/m2. Side effects were reversible and related to the vascular leak syndrome (VLS), i.e. decrease in serum albumin, edema, weight gain, hypotension, tachycardia, myalgia, and weakness. In all three patients receiving 20 mg/m2 NCI toxicity grade III was observed. Thus, 15 mg/m2 is the maximal tolerated dose (MTD) of RFT5-SMPT-dgA. 50% of the patients developed human anti-ricin A-chain antibodies (HARA) and/or human anti-mouse antibodies (HAMA). Clinical results included two partial remissions (PR), one minor response (MR), three stable disease (SD) and nine progressive disease (PD). In an extension of the phase I trial, five additional patients have been treated at the MTD.

To elucidate the effects of interferon-alpha (IFN-alpha) on normal human bone marrow in vivo, an immunomorphometric study was performed using trephine biopsy specimens without hematopoietic pathology. Samples were derived from patients with mycosis fungoides but no marrow involvement, who were undergoing low-dose IFN-alpha treatment. Parameters included density of reticulin (argyrophilic) fibers, CD61+ megakaryocytes, PGM1+ macrophages, the GSA-I lectin-expressing (activated) macrophage subpopulation, proliferative activity (PCNA staining), and apoptosis. Following IFN-alpha therapy (3 x 3 x 10(6) U/week between 6 and 21 months), morphometric evaluation of sequential bone marrow examinations revealed a significant increase in the number of megakaryocytes and the amount of reticulin fibers. Additionally, there was an overall decrease in PCNA+ cells, accompanied by a reduction in the incidence of apoptotic bodies. On the other hand, total number of macrophages and their activated subfraction remained unchanged. Opposed to in vitro findings, a fibrogenetic capacity of IFN-alpha associated with megakaryocyte growth was detectable. Moreover, contrasting with effects of IFN-alpha treatment in chronic myelogenous leukemia, the incidence of apoptosis was significantly reduced. This feature was assumed to contribute to a maintenance of steady-state hematopoiesis expressed by a nonaltered bone marrow cellularity in our specimens.

In the treatment of Hodgkin's disease (HD) remission rates of 80% have been achieved with combination regimens such as COPP/ABVD; 30%-50% of these patients relapse, however, and less than 25% of those in first relapse can be cured. Although 90% of adults with advanced Hodgkin's disease (HD) achieve a complete remission with new polychemotherapy regimens such as BEACOPP, it is too early to assess how many patients ultimately can be cured. In addition, these regimens are associated with severe side effects including infertility, cardiomyopathy or second malignancies. Thus, alternative strategies for improving the outcome of patients with HD have been developed. These approaches include new cytostatic drugs and biological agents. Here, we review the most recent developments including the new vinca alkaloid vinorelbine, the anthracycline idarubicin, the nitrogen mustard bendamustine, the recently developed nucleoside analogue gemcitabine, and immunotoxins against Hodgkin/Reed-Sternberg cells. We conclude that current polychemotherapy regimens could possibly be improved by introducing new agents with a different mechanism of action such as gemcitabine. In addition, some of these new drugs including gemcitabine or vinorelbine could contribute to the reduction of toxic side effects, thus resulting in an improved quality of life for patients with HD.

To improve the outcome for patients with relapsed or refractory multiple myeloma (MM), we combined idarubicin (Ida), dexamethasone and interferon (IFN) in a new regimen, 'I-Dexa'. Here we report our results of a phase I/II study using the I-Dexa protocol in an outpatient setting. A total of 31 patients were enrolled. Most patients were heavily pretreated with a median of two different chemotherapy regimen (range, 1-4). All but four patients had advanced disease (stage III). The dose of Ida was escalated to define the maximal tolerated dose (MTD) in this regimen. Four patients were treated with 5 mg/m2 and 27 patients with 7.5 mg/m2 Ida (day 1, i.v.). Dexamethasone was given at a fixed dose of 20 mg per os daily on days 2-5 and 11-14. Treatment was repeated at day 21 and consisted of up to six cycles. Patients who achieved a response or stable disease received IFN maintenance. IFN was administered three times weekly at a dose of 3 x 10(6) U/day s.c. until relapse. At the time of evaluation, 125 courses of I-Dexa were analyzed. The MTD of Ida in this regimen was 7.5 mg/m2. Hematological toxicity was mild including 5% leukocytopenia, 3% thrombocytopenia and 1% anemia (WHO grade III) at dose level 2. The MTD was defined by nonhematological toxicities including two WHO grade IV infections and one renal failure. The overall response rate including stable disease was 62.5% (PR: nine patients, 37.5%). So far, nine patients have been treated with IFN maintenance therapy with a median duration of 7 months (range, 1-16). In conclusion, I-Dexa can be given safely in an outpatient setting and is effective for the treatment of relapsed and refractory MM.

Idarubicin exhibits features rendering this drug unique among anthracyclines. The higher lipophilicity leads to faster accumulation in the nuclei, superior DNA-binding capacity and consequently greater cytotoxicity compared to daunorubicin. A major advantage over this reference drug is its ability to overcome MDR at least partially. Its major metabolite idarubicinol is as active as the parent compound and crosses the blood-brain barrier. Furthermore, idarubicin can be administered orally reaching sufficient plasma levels. Compared to other anthracyclines, the risk of cardiotoxicity as the main side effect for this group of drugs is reduced with idarubicin, when administered in a therapeutical dose. Thus, IDA has become an important drug in the treatment of acute leukemias and its potency in lymphomas, plasmocytomas and other solid tumors such as breast cancer is currently under investigation in several clinical studies.

We present a retrospective study using four different immunohistochemical markers (PCNA, Ki-67, 486p and p53) on paraffin sections from 104 selected cases with primary superficial transitional cell carcinomas of the bladder (59 cases pTa, 45 cases pT1, 40 cases G1, 64 cases G2). 53 of the 104 patients experienced recurrence of their bladder lesion, while 51 remained free of tumor. The distribution of staging, grading and multifocality was comparable in both groups of patients. Overall, the tumors that recurred had a significantly higher proportion of labeled cells for PCNA (p < or = 0.0001), Ki-67 (p < or = 0.006) and 486p (p < or = 0.0001). The latter antigen proved to be the most reliable marker. A less significant difference in staining pattern was found for p53 (p < or = 0.01). Evaluating the predictive value of the various antibodies separately for the groups with G1 vs. G2 carcinomas and pTa vs. pT1 tumors revealed a lower significance for all antibodies. The technique of immunostaining on paraffin sections facilitates further retrospective studies on archival material. These markers may provide additional information about the probability of recurrence of superficial bladder tumors. But at the moment they should only be utilized in selected cases.

Using an immunohistochemical method expression of PCNA, Ki-67, 486p and p53 antigen was investigated in paraffin sections of 119 patients with primary transitional cell carcinoma of the bladder. The purpose of this study was to detect recurring G1 and G2 bladder tumours compared with non-recurrent tumours. A relative large fraction of labelled cells for PCNA and Ki-67 was found in recurring G1 and G2 carcinomas. Moreover, recurrent transitional cell carcinomas showed a more positive staining pattern for 486p and p53, in contrast to non-recurrent carcinomas. Immunohistochemistry of these four antigens seems to yield additional information about the possibility of recurrence in G1 and G2 bladder carcinomas, thus allowing early, i.e. more aggressive, therapy.