Phase I-II study of epirubicin in multiple myeloma.

Forty patients with relapsed (26) or refractory (14) myeloma were treated with epirubicin of doses of 75, 90, 105, and 120 mg/m2 in groups of 6 or more patients to test for response, maximum tolerated dose, and toxicity. Thirteen patients had received prior doxorubicin and were included in the dose findings part of the study only. Staging was I (1), II (5), and III (34). Partial responses were seen in 5 patients (18.5%) (duration 1.5, 2, 2.5, 10, and 18 months) not previously treated with doxorubicin. No responses were seen in patients treated with prior anthracycline. Responses were not dependent upon dose level of epirubicin. Median nadir white blood cell count at the four-dose levels were 2,300, 1,000, 1,600, and 1,700/mm3 with median nadir granulocyte counts of 897, 720, 688, and 192/mm3. Fever/neutropenia was infrequently observed at the three lower dose levels but occurred in 6 of 10 patients at 120 mg/m2. Platelet nadirs were 110,000, 83,000, 169,000, and 42,000/mm3. Nonhematological toxicity was not dose dependent and included alopecia (100%), nausea/vomiting (40%), and stomatitis (25%). Six patients had greater than or equal to 0.10 changes in the resting ejection fraction with one patient developing congestive heart failure that responded to medical management. This patient had received prior doxorubicin and had a history of myocardial infarction. Epirubicin can produce remissions in patients with previously treated myeloma who have not received prior doxorubicin. Since the response rate was not enhanced at 120/m2 and since fever/neutropenia was seen regularly at this dose level, the recommended dose for further study is 105 mg/m2.

Constant infusion schedule for adriamycin: a phase I-II clinical trial of a 30-day schedule by ambulatory pump delivery system.

Eighteen patients received a continuous intravenous infusion of adriamycin for 14-60 days in a phase I study in which the dose rates were escalated from 2 mg/sq m/day to 5 mg/sq m/day to establish the optimal dose to be delivered over a 30-day period. The drug was delivered via a tunneled subclavian catheter by a portable infusion pump (Cormed model ML-6) primed to provide a volume of diluted drug of 10 cc/day. Leukopenia and stomatitis were observed at 4 mg/sq m/day doses or greater in 50% of courses. At doses less than 4 mg/sq m/day, only 3/17 courses (18%) were associated with stomatitis. Partial alopecia developed in all patients, but less than 50% of scalp hair was affected. The cumulative dose of continuous infusion adriamycin at 30 days is comparable to the dose delivered by standard bolus intermittent schedules (60-90 mg/sq m g 21 days), but the adverse drug effects are eliminated or substantially reduced. Cardiac toxicity was assessed in selected patients treated to 450 mg/sq m or greater by cardiac biopsy and/or gated pool studies. No histopathologic lesions were noted in 3 patients receiving 450 mg/sq m or greater. The recommended daily dose rate of adriamycin in this protracted infusion regimen is 3 mg/sq m/day. The phase II study of this schedule and dose rate in 38 additional patients (a total of 52 evaluable patients) demonstrated objective responses in 1/9 soft tissue sarcoma, 1/3 mesothelioma, 1/3 hepatoma, and 2/13 breast cancer. Phase III studies of the protracted continuous infusion schedule for adriamycin are indicated in that clinical activity is demonstrated at a substantial reduction in toxicity. Pharmacologic studies expanding the existing data base are also necessary.

Properties of the blood group LW glycoprotein and preliminary comparison with Rh proteins.

The major component immunoprecipitated from human red cell membranes by murine monoclonal antibodies (BS46 and BS56) against the LW blood group antigens is a 42,000 mol. wt glycoprotein. Upon digestion by an N-glycanase the LW component migrated as a 25,000 mol. wt component on SDS gels, whereas treatment by an O-glycanase led only to a small size reduction (2000). These data suggest that the LW glycoprotein might carry approximately eight to nine N-linked sugar chains and only a few (one or two) O-linked oligosaccharide chains. A minor component of 31,000 mol. wt was also identified in the LW immunoprecipitate. Preliminary analyses by two-dimensional peptide mapping indicate that the 31,000 mol. wt polypeptide is identical to authentic Rh proteins, therefore raising the possibility that the Rh and LW antigens are associated in the membrane as a functional complex called Rh cluster. Since the N-deglycosylated form of the LW and RhD proteins have different sizes (25,000 vs 31,000-32,000 respectively) and since their externally 125I-labelled domains have different two-dimensional peptide maps, it is concluded that LW is probably not a simple glycosylated form of the Rh proteins.

Paclitaxel, cisplatin and etoposide combination chemotherapy: a comparison of dose intensity in two multifractionated dose schemas.

66 patients with a variety of tumour types received the multifractionated TPE three drug regimen in a non-random allocation as a 5 day schedule (schedule A) or as a twice weekly schedule (schedule B). The dose per fraction for each component drug was 35, 40 or 50 mg/m2 for both paclitaxel and etoposide and for cisplatin, the dose per fraction was 15 mg/m2. The total paclitaxel and etoposide dose was 175, 200, 250 mg/m2 3 week cycle. For schedule A, grade 3 or 4 neutropenia was observed in 70/114 cycles (61%) with two treatment related deaths from 50 treated patients. For schedule B, grade 3 neutropenia was observed in 1 of 30 courses (3%) with one drug related death from 19 treated patients. Dose intensity was increased by 20% for both paclitaxel and etoposide with the twice weekly schedule and at all dose levels, with haematological toxicity substantially reduced relative to schedule A. Using multifractionated schedules, a twice weekly open ended schedule results in an approximately 20% greater dose intensity and less toxicity compared with a 5 day schedule repeated every 3 weeks. The recommended dose schedule for TPE is paclitaxel 40 mg/m2; cisplatin 15 mg/m2 and etoposide 40 mg/m2 twice weekly for 3 weeks repeated every 4 weeks.

Coincidence of Epstein-Barr virus reactivation, cytomegalovirus infection, and rejection episodes in renal transplant recipients.

Reactivation of the Epstein-Barr virus was reported to occur frequently under immunosuppressive therapy following organ transplantation. However, little is known about the clinical significance of these EBV reactivations. Therefore, we searched for correlations among the treatment with various immunosuppressive drugs, the incidence of CMV infections, rejection crises, and serological signs of EBV reactivation. EBV-specific antibodies were measured with novel ELISAs, utilizing the recombinant antigens p72 (for anti-EBV nuclear antigen [EBNA]1-IgG), p54, and p138 (anti-early antigen [EA]-IgM, -IgG, -IgA) in a follow-up study of 79 renal transplant recipients. Patients receiving antithymocyte globulin or antilymphocyte globulin therapy showed increasing anti-EA-IgG and -IgA more often than did patients not receiving antithymocyte globulin or antilymphocyte globulin therapy (P < 0.05). In patients receiving OKT3 antirejection therapy, anti-EA-IgM seroconversion was found more frequently (P < 0.01). A significant correlation was also found between groups of patients who had had at least one rejection episode versus patients without any sign of organ rejection, and the incidence of increasing anti-EA-IgG (P < 0.05). Since in most of these patients signs of EBV reactivation followed the appearance of the rejection episode, this may not be due to viral-induced rejection but may be caused by the reinforced immunosuppression during antirejection therapy. As opposed to patients with no signs of CMV infection and with nonsymptomatic CMV infection, patients undergoing symptomatic CMV infection showed anti-EA-IgM seroconversion (P < 0.01), increasing anti-EA-IgA (P < 0.01), and decreasing anti-EBNA-IgG (P < 0.01) more frequently. Our results confirm the role of immunosuppressive therapy in the pathogenesis of EBV reactivation. We further demonstrate a striking coincidence of EBV reactivation and symptomatic CMV infection.

Inhibition of tumor growth in a mouse fibrosarcoma after interleukin 2 application.

Highly purified natural IL-2, obtained from induced human PBL's, was used to treat tumor-bearing mice. 3 X 10(6) vital cells of chemically induced fibrosarcoma BALB/c female Meth A were transplanted subcutaneously to female mice. Repeated injections of 10,000 U IL-2 into established tumors of 7-8 mm diameter led to complete rejections in 75% of the treated animals. Single intratumoral injections and other routes of the application of IL-2 did not significantly influence the tumor growth rate.

Serological diagnosis of infectious mononucleosis using three anti-Epstein-Barr virus recombinant ELISAs.

A new Epstein-Barr virus (EBV) ELISA system (Biotest Anti-EBV recombinant) was evaluated for usefulness for routine diagnosis of EBV primary infection. The assay system is composed of three different microtest plates coated with three highly purified recombinant EBV antigens. The early antigens p138 (BALF2, truncated) and p54 (BMRF1, whole sequence) are used as a mixture for testing IgM (assay 1) and IgG (assay 2) antibodies. In addition, the EBNA-1 antigen p72 (BKRF1, carboxy-half) is used for detecting IgG antibodies (assay 3). Three panels of sera were examined in direct comparison with standard immunofluorescence (IF): Specimens of (i) 120 infectious mononucleosis (IM) patients, (ii) 60 patients with acute CMV infection, toxoplasmosis or rheumatic disease, respectively, and (iii) 185 healthy blood donors as a control group. 119 IM patients were clearly recognized as having acute primary infection (sensitivity 99.2% compared to VCA-IgM by IF). Three apparently false-positive results were obtained with patients of other diseases and none within the control group (specificity 98.8%). The data suggest that the recombinant ELISA can be used advantageously for standardized rapid diagnosis of acute EBV primary infection.

IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins.

Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (p52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (pp150, aa 994-1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELISA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection.

Streptozotocin for metastatic malignant melanoma.

Streptozotocin was administered at a dose of 500 mg/m2/day by continuous infusion for 120 hours to 14 patients with advanced malignant melanoma. No responses were observed in this group of patients and the median survival for the entire group was 2 months. At the dose and schedule delivered, streptozotocin is inactive against malignant melanoma.

Adriamycin, cyclophosphamide, and etoposide (VP-16-213) in extensive-stage small cell lung cancer.

A three-drug regimen composed of adriamycin, 50 mg/m2 and cyclophosphamide, 500 mg/m2 administered on day 1; and VP-16-213, 50 mg/m2 days 1-5, with courses repeated at 3-week intervals, was studied in 24 consecutive patients with extensive-stage small cell lung cancer (SCLC). Twelve of 33 patients (36%) evaluable for toxicity developed life-threatening marrow suppression and 12% died of septicemia following the first course of treatment. Eleven of 24 patients (46%) with extensive disease achieved an objective response and only one was classified as a complete response. Survival was related to performance status and metastatic site but was not influenced by tumor response. The present study is distinctive from that of previous reports of the same or similar three-drug regimen in that the response rate is lower and toxicity is substantial. Nonetheless, survival as measured by median duration (7.9 months) and proportion alive at 1 year (35%) is comparable to that of previous reports.

Cancer chemotherapy via ambulatory infusion pump.

One hundred twenty-four patients with metastatic malignancy were treated with four different single agent infusion programs by constant intravenous infusion for 30 or more days. Drug was administered via a tunneled subclavian line by a battery-driven peristaltic pump (Cormed model ML6) on an ambulatory basis. This schedule allowed for increased cumulative drug dose for 5-Fu, decreased tolerated dose for vinblastine, and comparable doses for adriamycin and mitomycin-C relative to that delivered with the standard intermittent bolus schedule. Therapeutic effects were observed for three of four drugs studied: 5-Fu 13/31 colorectal cancer; adriamycin 7/29; and vinblastine 4/12, including 2/4 melanoma. Adverse effects were significantly reduced, particularly with regard to gastrointestinal toxicity, but also in adriamycin-associated cardiac effects and hair loss. Phase III comparative trials of intermittent bolus therapy with protracted infusion therapy are in progress for 5-Fu in advanced colorectal cancer and for adriamycin in specific tumors. Ambulatory pump infusion (API) chemotherapy is technically feasible and has improved patient tolerance to chemotherapy while demonstrating similar, if not comparable, antitumor effects.

Phase II study of aclarubicin in acute myeloblastic leukemia.

Aclarubicin is a new anthracycline antibiotic that produces substantially less cardiotoxicity in animals that does doxorubicin. Based upon prior Phase I and II trials in leukemia, a Phase II study in acute myeloblastic leukemia was developed to assess the response rate and toxicity in previously treated patients. Forty patients received aclarubicin 100 mg/m2 per day X 3 with repeated course on days 14-16 if marrow hypoplasia was not produced. Complete responses were achieved in 27.5% (11/40) with durations of 1.5, 2, 2, 2, 3, 3+, 4, 5+, 32+, 33+, and 34+ months. Toxic effects of this therapy included severe neutropenia and thrombocytopenia, nausea/vomiting, mucositis, and diarrhea. No patient developed significant changes in the left ventricular ejection fraction, as measured by radionuclide angiography, or any clinical cardiac symptoms. Alopecia was minimal. Aclarubicin can produce a significant response rate in previously treated patients with acute myeloblastic leukemia and should be considered for study in initial therapy.

From IgG monoclonals to IgM-like molecules.

One problem in blood group testing is that IgG monoclonal antibodies, in contrast to IgM, do not usually agglutinate erythrocytes. One of the reasons is the high zeta potential induced by the negative charge of the cell surface. During the last few years, we have produced a series of human monoclonal antibodies by the conventional fusion technique directed against antigens of the Rh blood group system. Some of these monoclonals, especially those directed against Rh-subgroups such as the c-antigen, were mainly of the IgG-subtype and unsuitable for agglutination tests. We have therefore tried to establish a molecular biological method to make IgM-like molecules from IgG monoclonals. From the c-antigen specific human hybridoma BS 240 (IgG subtype), we isolated mRNA that was transcribed into cDNA and then amplified by PCR using family specific primers. The heavy and light chain products were cloned into the pHen vector containing a DNA linker fragment, a myc-tag for identification and a His-tag for purification. After transformation in E.coli and phage rescue with helper phage, the culture supernatant was screened for antigen positive recombinant phage antibodies as a first control for specificity using c-antigen positive erythrocytes and anti-M13 antibodies as bridging antibodies (Coombs technique). Erythrocytes being negative for the c-antigen served as a negative control. After changing the culture conditions, soluble single chain fragments (scFv) were obtained from the periplasmatic extract. Specificity was shown using the c-antigen positive and negative erythrocytes and the 9E10 antibody (anti-myc) as a bridging antibody. To obtain IgM-like molecules, DNA coding for the specific scFv was cloned into the vector pSTE containing DNA coding for the monomer of core streptavidin. After expression, purification and refolding of the monomer, the core streptavidin combines to form tetrameric structures, termed scFv::strep, that are able to bind biotin as shown using ELISA plates coated with biotinylated BSA. Binding was detected with 9E10 and a peroxidase conjugated secondary antibody. In the agglutination assay, the construct was able to agglutinate c-antigen positive erythrocytes but not the negative erythrocytes. These experiments show that it is possible to construct IgM-like agglutinating molecules from cells containing secreting IgG antibodies. Experiments employing human antibody libraries instead of hybridoma cell lines are now in progress.

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' analysis.

A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library. A repetitive sequence of 27 bp length was identified which is contained in the probe approx. 40 times. The MZ 1.3 repeat shows variable homology of 53-73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome. Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI/Pvu II, and Rsa I. An average of 18 polymorphic fragments was observed using Hinf I as enzyme. The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 +/- 7.2%. An example for the application of MZ 1.3 to paternity testing in an incest case is given. The probe can be used with radioactive or non-radioactive detection systems. An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.

Flow cytometric investigation of non-specific erythrocyte antigens.

Thirteen monoclonal antibodies submitted to the Third Workshop on Erythrocyte Antigens from the panel "non-specific erythrocyte antigens" were tested for their reactivity with different types of cells. Most of them were defined as specific for adhesion antigens. The CD 44 antibodies 2D3-1, 2D3-2, 2D3-3 and 2D3-4 reacted as expected for CD 44 except their negative reactivity with the myeloid cell line HL 60 and B-cell line Raji. The CD 47 antibodies 2D3-5 and 2D3-6 reacted specific. Only with Raji and T-cell line MOLT 4 the CD 58 antibodies 2D3-7 and 2D3-8 showed reactivity as expected which indicates that they are "CD 58 related". The CD 99 antibody 2D3-9 shows similar results as expected for a CD 99 specific antibody except its high reactivity against Raji. From the RBC-related antibodies 2D3-11 and 2D3-12 the latter becomes completely negative with trypsin treated erythrocytes. The antibody is negative on normal peripheral blood lymphocytes but reacts with transformed cell lines like Raji and MOLT 4. With a view to their reactivity to the cells tested at least 2D3-13 of the Rh-related antibodies seems to be similar to CD 47 antibodies.

A structural model for 30 Rh D epitopes based on serological and DNA sequence data from partial D phenotypes.

Both cDNA RHD sequences and reactivity with monoclonal anti-D have been reported in a number of partial D phenotypes, where parts (some epitopes) of the normal D antigen are missing, and anti-D of restricted specificity may be made in response to challenge with normal D positive blood. This paper analyses these reports together and proposes a model for the structure which comprise the epitopes of the Rh D antigen. Some epitopes are proposed to be comprised of continuous peptide sequence within one extracellular loop, whereas others require interactions between two or the extracellular peptide loops.

Generation of monoclonal antibodies directed against the immunogenic glycoprotein K8.1 of human herpesvirus 8.

Human Herpesvirus 8 (HHV-8) is clearly associated with Kaposi's sarcoma (KS), body cavity-based lymphomas (BCBL), and certain forms of multifocal Castleman's disease (MCD). It appears to be the sexually transmissible agent involved in the development of AIDS-associated KS. HHV-8 genomes are invariably present in BCBL-derived cell lines where lytic replication of the virus can be induced by phorbol esters (PE). First-generation HHV-8 serological assays were based on these cell lines. More recently, several genes encoding HHV-8 antigens have been identified. One of the most reactive antigens is encoded by HHV-8 open reading frame K8.1. Although K8.1 does not exhibit overt sequence homology to any other known gene, it is likely to be analogous to gp220/350 of Epstein-Barr or gp150 of murine herpesvirus-68, virion-envelope glycoproteins involved in target cell recognition. Mice were immunized with purified GST-K8.1 fusion protein expressed in E. coli. After fusion of murine plasma cells with the myeloma cell line P3-X63-Ag8. monoclonal antibodies (MAbs) were generated, which are specifically directed against K8.1 protein. The binding site for each MAb was identified by deletion mutant analysis using recombinant GST-K8.1 mutants and K8.1-specific peptides. Without exception, the epitopes recognized by these MAbs were located within the N-terminal part of the protein [amino acids (aa) 29 to 80], thus identifying a highly immunogenic region. These antibodies will not only be useful tools for HHV-8 diagnostics, but will also facilitate the analysis of K8.1 function.

Biotechnology in transfusion medicine.

In an overview, this paper summarizes the new possibilities based on biotechnology, such as monoclonal antibodies and genetic engineering. Examples are given where these new techniques are already used in transfusion work. In addition, some perspectives for the future in relation to these techniques are given.

[Molecular biology and serology in the ABO system]. / Molekularbiologie und Serologie im AB0-System.

Using high-affinity monoclonal anti-A und anti-B antibodies the B(A) and A1(B) phenomena were detected. This is due to the high similarity of the final transferases adding the A- and B-specific sugars. In the meantime these proteins have been cloned and the biochemistry explains the serological findings. In addition, also for the AB0 subgroups transferases of different activities have been found and cloned, giving also here the answer to the differences between A1 and A2.

Monoclonal antibodies against red blood cell antigens used in routine practice and research.

This paper describes the application of monoclonal antibodies (mAbs) in routine practice and research. Carefully selected anti-A and anti-B mAbs show stronger reactions, especially when the antigen density is diminished, detect better the weak variants like Ax and Bw, do not react with acquired B, and do not express anti-T activity. For the MN system murine mAbs are used successfully since several years in routine practice as well as for testing the Lewis antigens. They are superior to the polyclonal reagents. In the Rh system the mAbs are now going to be established. The reactivity of mAbs with weak D red blood cells and so-called categories is discussed. The paper gives examples for the use of mAbs as a very important tool for red blood cell antigen research leading to a new understanding of genetics, biosynthesis, and molecular structure of the red cell antigens.