Nonlethal transfusion associated graft-versus-host disease in a severe combined immunodeficient patient.

An X-linked severe combined immunodeficient (SCID) patient received a nonirradiated erythrocyte transfusion and developed transfusion-associated graft-versus-host disease (TAGVHD), which was controllable with high-dose corticosteroids. Haplo-identical SCT was performed, after a myeloablative conditioning regimen. At day +26, he developed GVHD. Chimerism studies revealed DNA of the erythrocyte transfusion donor (ETD) and recipient only. Because of early nonengraftment and the presence of alloreactive T cells of ETD origin, the patient was treated with an immunosuppressive conditioning regimen followed by a second SCT from the same donor. While tapering immunosuppression, he again developed mild GVHD, and DNA of ETD and bone marrow donor origin were both present. On cyclosporin, the ETD-DNA signal finally disappeared. High-resolution HLA typing revealed haplo-identity between BMD, ETD and the patient, which might have contributed to the relative mild course of the TAGVHD.

Evaluation of automated nucleic acid extraction devices for application in HCV NAT.

BACKGROUND: To further improve the safety of the blood supply, various national blood transfusion organizations presently use or are in the process of implementing routine HCV NAT in minipools. According to the Committee for Proprietary Medicinal Products (CPMP) of the European Union, the HCV NAT detection limit of the assay should be 100 IU per mL (270 geq/mL) for testing initial plasma pools. Paul Ehrlich Institute (PEI) regulations stipulate that 5000 IU per mL (13,500 geq/mL) must be detected to calculate the amount contributed by individual donations composing the minipool. The sensitivity for HCV RNA extraction achieved by three commercially available laboratory kits was compared. STUDY DESIGN AND METHODS: Nucleic acids from 1-in-3 serial dilutions of an HCV RNA run control (Pelispy, CLB) were extracted with three kits (Cobas Amplicor, Roche Diagnostic Systems; BioRobot 9604, Qiagen; and NucliSens Extractor, Organon Teknika). HCV PCR of all extracts was performed using a second-generation Cobas Amplicor HCV test and the Cobas Amplicor analyzer. RESULTS: The manual Cobas Amplicor, the BioRobot 9604, and the NucliSens Extractor setups allow a 95-percent HCV RNA detection limit of 129, 82, and 12 geq per mL, respectively. The maximal pool size for the manual Cobas Amplicor, the BioRobot 9604, and the NucliSens Extractor kits that would still meet the PEI criteria for HCV NAT in minipools was calculated at 104, 164, and 1125 donations, respectively. CONCLUSION: All three HCV NAT kits evaluated meet the criteria set by CPMP and PEI. The highest sensitivity for HCV NAT screening can be achieved with the high-volume NucliSens Extractor method in combination with the Cobas Amplicor HCV v2.0 test on the Cobas Amplicor analyzer.

A simple strategy to look back on posttransfusion hepatitis B in a multitransfused patient.

BACKGROUND AND OBJECTIVES: In January 1996, a case of hepatitis B virus (HBV) seroconversion in a multitransfused patient was reported to the blood bank From March through October 1995, the patient had received 23 units of red cells and 30 units of pooled platelet concentrates, encompassing an exposure to a total of 200 whole blood donations. MATERIALS AND METHODS: In order to trace hepatitis B surface antigen (HBsAg)-negative but HBV-infectious blood donation(s), we tested samples of the donors obtained > or = 3 months after the implicated donations for anti-HBc (Corezyme EIA, Abbott). From 172/200 donors, archived samples of subsequent donations were available for this purpose. The remaining 28 donors were reinvited to the blood bank to obtain an additional blood sample for anti-HBc testing. RESULTS: 1/200 follow-up donor samples was anti-HBc-positive. Retrospective testing of the implicated HBsAg-negative blood donation of this donor revealed anti-HBc-negative and HBV-DNA-positive results. The patient was transfused with the platelets of the HBV-infectious donation. On looking back, the other blood products prepared from this HBV-infectious donation caused posttransfusion HBV infection (PT-HBV) in 2 additional patients. CONCLUSION: Anti-HBc testing on mainly archived follow-up samples of 200 donors implicated in PT-HBV was a rapid, simple cost-effective and donor-friendly method to identify an infectious but HBsAg-negative, anti-HBc-negative and HBV-DNA PCR-positive blood donation. Routine anti-HBc screening would not have prevented this HBV transmission.

New hepatitis B virus mutant form in a blood donor that is undetectable in several hepatitis B surface antigen screening assays.

BACKGROUND: Envelope mutant forms of hepatitis B virus (HBV), impairing HBV antibody recognition, have been reported with mutations in single or multiple sites of the hepatitis B surface antigen (HBsAg) group-specific "a" determinant. Blood donors infected with such an HBsAg mutant form of HBV may escape detection by HBsAg screening assays and therefore may affect the safety of the blood supply. CASE REPORT: A repeat blood donor became HBsAg-reactive in an enzyme immunoassay. Confirmatory testing yielded negative results for HBsAg in a radioimmunoassay and in four enzyme immunoassays used in blood donor screening. The specificity of the HBsAg reactivity in the first enzyme immunoassay was confirmed by HBsAg neutralization with antibody to HBsAg. Additional HBV confirmatory test results were positive for antibody to hepatitis B core antigen and antibody to hepatitis B e antigen; negative for antibody to HBsAg and for hepatitis B e antigen; and positive for HBV DNA. DNA sequence analysis of the "a" determinant region of HBsAg revealed amino acid substitutions from Q (Gln) to R (Arg) at codon 129 and from M (Met) to T (Thr) at codon 133. CONCLUSION: This case illustrates the presence of HBsAg mutant forms of HBV in a West European blood donor population that were undetected by several HBsAg screening assays. Adaptation of HBsAg screening is indicated to overcome deficiencies in sensitivity in detecting HBsAg mutant forms of HBV. Screening for antibody to hepatitis B core antigen or HBV DNA may also detect blood donors infected with HBsAg mutant forms of HBV

Optimization of purging of autologous bone marrow grafts for children with precursor B acute lymphoblastic leukemia.

In our laboratory, a two-step procedure is used for purging precursor B ALL from autologous bone marrow grafts of children in second bone marrow remission. An immunorosette depletion method with CD19 and CD22 MAbs is followed by one cycle of complement-mediated cell lysis with CD9 and CD10 MAbs. The aim of the present study was to determine if the efficacy of this procedure could be further enhanced by including CD20 and CD72 MAbs in the current protocol. Leukemia-contaminated remission bone marrow was simulated by mixing cell line cells and normal bone marrow cells. The efficacy of purging of malignant cells was determined by culturing the cells in a limiting dilution assay. The effect of including CD20 and CD72 in the immunorosette depletion was limited. In contrast, when these MAbs were added during complement-mediated cell lysis, a significant increase in depletion of tumor cells was observed. This was true when complement lysis was carried out alone (0.4 versus 3.0 log depletion for Ros cells) and when it was preceded by immunorosette depletion (2.7 versus 4.1 log depletion for Ros cells). The loss of hematopoietic progenitor cells was not greater than with the current purging protocol.

Human immunodeficiency virus (HIV) antibodies detected by new assays that are enhanced for HIV-1 subtype O.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) subtype O infections are not reliably detected by commonly used anti-HIV-1/2 screening assays. Therefore, anti-HIV-1/2 assays have been modified to increase their sensitivity in detecting antibodies to HIV-1 subtype O. STUDY DESIGN AND METHODS: Two new anti-HIV-1/2 enzyme-linked immunosorbent assays (ELISAs) (Abbott Plus and Ortho Enhanced) were compared with a currently used anti-HIV-1/2 ELISA (Abbott Recombinant) in various serum panels: 91 Western blot-confirmed anti-HIV-1-positive samples, 20 samples from Western blot-confirmed HIV-1-infected patients in log3 serial dilutions, and 1463 samples from consecutive, volunteer, nonremunerated blood donors. RESULTS: Among 91 anti-HIV-1 Western blot-positive samples, 2 (2.2%) were missed by the Abbott Recombinant ELISA, but all 91 were detected by the Abbott Plus and Ortho Enhanced ELISAs. In contrast, two discrepant samples were found to react in viral lysate-based assays. In serial dilutions, Ortho Enhanced ELISA was significantly less sensitive than the Abbott Recombinant and Abbott Plus ELISAs, with the latter two being of comparable sensitivity. The specificities of Abbott Recombinant, Abbott Plus, and Ortho Enhanced ELISAs in 1463 blood donors were 100, 99.93, and 99.86 percent, respectively. Routine testing of 29,102 donations with the enhanced Abbott Plus ELISA revealed a specificity of 99.93 percent. CONCLUSION: Two Western blot-confirmed anti-HIV-1-positive samples were missed by the Abbott Recombinant ELISA but detected by the Abbott Plus and Ortho Enhanced ELISAs. The analytic sensitivity of the Ortho Enhanced ELISA was inferior to that of both Abbott ELISAs. The specificities of the Abbott Recombinant, Abbott Plus, and Ortho Enhanced ELISAs were comparable.

Rearrangement status of the malignant cell determines type of secondary IgH rearrangement (V-replacement or V to DJ joining) in childhood B precursor acute lymphoblastic leukemia.

Immunoglobulin heavy chain (IgH) oligoclonality in childhood B precursor acute lymphoblastic leukemia (ALL) as determined by Southern analysis is found in 30-50% of patients and has been shown to be the result of ongoing IgH rearrangement (mostly V(H)-replacement and V(H) to D-J(H) joining) after malignant transformation. It is unknown however, what determines the type of secondary rearrangement. Also the biological basis of the variable degree of oligoclonality observed in childhood ALL is poorly understood. We analyzed in detail the IgH rearrangement status of the leukemic cells for a random panel of 18 childhood B precursor ALL patients by polymerase chain reaction (PCR)/sequencing analysis and by Southern analysis. By Southern analysis 10/18 (55.6%) patients were considered oligoclonal and 8/18 (44.4%) monoclonal. In contrast, by PCR minor clonal rearrangements were detected in 14/18 (77.8%) patients. V(H)-replacement was found in 7/14 patients, V(H) to D-J(H) joining in 6/14 patients and an unusual type of secondary rearrangement, V(H)-D to J(H) joining, in one patient. Only a single type of secondary rearrangement was detected in each patient. The type of secondary rearrangement (V(H)-replacement or V(H) to D-J(H) joining) depended on the rearrangement status (VDJ/VDJ or VDJ/DJ, respectively) of the dominant leukemic clone as determined by Southern analysis. We found that in addition to a more 'advanced' IgH rearrangement status patients with V(H)-replacements also have a more 'advanced' TCRdelta rearrangement status, which possibly reflects exposure of both the IgH locus and the TCRdelta locus to recombinase activity in a preleukemic clone. Finally, we investigated a putative relationship between oligoclonality by Southern analysis and S-phase fraction of the leukemic cell population. We found a significantly lower percentage cells in S-phase for oligoclonal patients as compared to monoclonal patients. Our data add to the understanding of ongoing rearrangement of antigen receptor loci in childhood ALL and have implications for the monitoring of minimal residual disease by PCR.

PCR-positivity in harvested bone marrow predicts relapse after transplantation with autologous purged bone marrow in children in second remission of precursor B-cell acute leukaemia.

Purging of autologous bone marrow (BM) grafts of children in second remission after a relapse of precursor B acute lymphoblastic leukaemia (ALL) in the BM has been carried out in our laboratory since 1987, initially by complement mediated cell lysis. This protocol was extended by performing an immunorosette depletion before lysis with complement. The aim of the present study was to assess by polymerase chain reaction the presence of residual leukaemic cells in the BM grafts before and after purging. The results were then correlated to clinical outcome. In 24/28 patients a PCR product was obtained by amplification of IgH and/or TcR junctional regions. BM before purging was available for analysis in 13 patients. We found that leukaemic cells could be detected in 8/13 (62%) of these grafts before purging . All these eight patients experienced a relapse, regardless of whether the purging procedure had been successful (defined as achievement of PCR-negativity) or not. In contrast, none of the five patients with PCR-negative grafts before purging relapsed (P = 0.0008). One patient died due to transplant-related toxicity. Of the remaining 23 patients, nine patients received a PCR-positive BM graft after purging. All these nine patients experienced a relapse as compared to 6/14 whose BM was PCR-negative after purging (P = 0.0072). Two of eight PCR-positive BM grafts could be purged to PCR-negativity. Thus, improvements both in treatment of leukaemia and in purging efficacy are still needed.

Clonal evolution of immunoglobulin heavy chain rearrangements in childhood B-precursor acute lymphoblastic leukemia after engraftment in SCID mice.

We grafted childhood B-precursor acute lymphoblastic leukemia (ALL) bone marrow (BM) cells into mice with severe combined immunodeficiency (SCID), in order to study the clonal evolution of immunoglobulin heavy chain (IgH) rearrangements in the absence of selective pressure by chemotherapy. BM cells from nine patients (six diagnosis samples and three relapse samples) were intravenously injected into SCID mice (three mice for each patient). All mice injected with cells from four patients developed a leukemia-like illness 12-40 weeks after injection. By PCR, new subclones that were the result of ongoing IgH rearrangement according to the mechanism operative in the injected cell populations (VH-replacement or VH to D-JH joining) were detected in the engrafted cell populations for all four patients. Subclones were mouse-specific, suggesting that subclone formation is a continuous process. Southern analysis after engraftment was unaltered as compared to the injected cells for one patient and revealed changes indicative of altered clonal composition for three patients. For two patients the observed changes possibly reflect the initial engraftment of a limited number of cells and occurred without changes in other parameters of the engrafted cell population, such as time needed for the development of leukemia, macroscopic organ involvement, immunophenotype and S-phase fraction. In one patient, we demonstrated the selective outgrowth of only a single cell type present at diagnosis, as characterized by IgH rearrangements. Our data show that evolution of clonal IgH rearrangements in B-precursor ALL may occur without the selective pressure of chemotherapy. Additionally, in some patients subclones present at diagnosis, as defined by IgH rearrangements, also possess different biological properties.

Treatment of childhood Hodgkin's disease with ABVD without radiotherapy.

Seventeen previously untreated children with Hodgkin's disease were treated with six courses of the combination adriamycin, bleomycin, vinblastine, and DTIC (ABVD), without radiotherapy, from 1984-1987. In all patients, complete remission was attained. After a median follow-up period of 73.5 months (range 59-98 months) five patients had a relapse after 4, 5, 11, 21, and 34 months, respectively, from attainment of complete remission. In 12 patients with stages I and II, two relapses occurred. Three out of five patients with stage III and stage IV developed a relapse. Based upon these results, we conclude that ABVD might be an appropriate treatment for newly diagnosed children with Hodgkin's disease stages I and II. However, for children with stages III and IV more intensive treatment is needed. Radio-therapy should be withheld for children with refractory disease, residual disease, or relapse.

High cure rate with a moderately intensive treatment regimen in non-high-risk childhood acute lymphoblastic leukemia. Results of protocol ALL VI from the Dutch Childhood Leukemia Study Group.

PURPOSE: Here we report the results of a nationwide cooperative study in the Netherlands on acute lymphoblastic leukemia (ALL) in children. The aim of the study was to improve the cure rate and to minimize side effects in a group of non-high-risk ALL patients, especially with regard to the CNS. A second aim was to study potential prognostic factors. METHODS: Children (age 0 to 15 years) with non-high-risk ALL (WBC count < 50 x 10(9)/L, no mediastinal mass, no B-cell phenotype, and no CNS involvement) were treated with a uniform protocol, ALL VI. The treatment protocol used 6-week induction regimen with three drugs (vincristine, dexamethasone, and asparaginase), three weekly doses of intravenous (IV) medium high-dose methotrexate (2 g/m2), and 2-year maintenance therapy that consisted of alternating 5-week periods of methotrexate and mercaptopurine and 2-week periods of vincristine and dexamethasone. In the first year of maintenance, triple intrathecal therapy was administered every 7 weeks. RESULTS: From December 1, 1984 until July 1, 1988, 291 children with ALL were diagnosed; 206 were categorized as non-high-risk (71%), and 190 were treated according to protocol ALL VI. At 8 years, the event-free survival (EFS) rate was 81% (SE = 3%) and survival rate 85% (SE = 2.9%); the median follow-up time was 7.3 years (range, 36 to 117 months). The CNS relapse rate was 1.1% (two of 184 patients who achieved a complete remission [CR]). The only factor found to be of negative prognostic importance in terms of EFS (P = .05) was a positive acid phosphatase reaction. CONCLUSION: For children with non-high-risk ALL, the combination of IV medium high-dose methotrexate (2 g/m2 times three), triple intrathecal therapy in the first year of maintenance treatment, and the use of dexamethasone for induction and pulses during maintenance treatment has proved to be highly effective, especially in the prevention of CNS relapse. A high cure rate was achieved without the use of anthracyclines, alkylating agents, and cranial irradiation.

Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia.

In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10+ CD19+ cells and CD20- CD22+ cells in the CD34+ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10-28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM sample indicated that only a subpopulation of the CD34+ CD10+ CD19+ and CD34+ CD20- CD22+ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted.

[Children with acute lymphoblastic leukemia: prognostic significance of polymerase-chain-reaction analysis of minimal residual disease]. / Kinderen met acute lymfoblastenleukemie: de prognostische betekenis van analyse door middel van de polymerase-kettingreactie van minimale residuale ziekte.

OBJECTIVE: Determination of the usefulness of polymerase chain reaction (PCR) for the detection of minimal residual disease (MRD) in bone marrow in children suffering from progenitor-B cell acute lymphoblastic leukaemia during and after treatment. DESIGN: Descriptive. SETTING: Emma's Children's Hospital, Academic Medical Centre, University of Amsterdam and Central Laboratory of the Dutch Red Cross, Amsterdam. METHOD: Of 50 children suffering from progenitor-B cell ALL, stored bone marrow samples and bone marrow slides were investigated: 328 bone marrow samples were analysed by PCR for IgH/TCR delta; 34 patients were analysed at the end of induction therapy. Follow-up period was 20 to 133 months. RESULTS: Twenty-two patients stayed in continuous complete remission (CCR), 28 patients experienced a recurrence (REC). Reduction of tumour mass was higher in the CCR group. At the end of induction therapy 2/18 CCR patients and 10/16 REC patients were PCR positive (p = 0.005). PCR positivity was not related with known prognostic factors. After recurrence 6/8 patients, who became PCR negative, stayed in remission. All patients who stayed positive after treatment for their recurrence died from leukaemia (p = 0.006). All children who were only temporary PCR negative suffered a recurrence. CONCLUSION: Analysis of MRD by means of PCR on bone marrow samples during and after treatment for progenitor-B cell ALL is of prognostic importance.

Degradation of radioiodinated B cell monoclonal antibodies: inhibition via a FCgamma-receptor-II-mediated mechanism and by drugs.

Our aim is to treat patients with B cell malignancies with radioimmunotherapy using monoclonal antibodies (mAb) such as CD19, CD20 and CD22. In this study we investigated the rate of internalization and catabolism of these mAb. After 24 h at 37 degrees C, 20%-25% of initially cell-bound (125)I-CD19 mAb and (125)I-CD22 mAb was degraded in B cells, whereas almost no degredation occurred after binding of (125)I-CD20 mAb. For B cells expressing Fcgamma receptor II (FcgammaRII), isotype-dependent degradation was noted as the CD19 IgGl mAb showed an enhanced degradation rate compared to the switch variant IgG2a. The effect of various pharmaceutical agents that delay the internalization or subsequent degradation of mAb was evaluated. The degradation was inhibited most effectively by a combination of etoposide and vinblastine, resulting in accumulation of radioactivity in the target cell. Also the simultaneous application of CD20 or CD22 with (125)I-CD19 mAb or of CD20 with (125)I-CD22 mAb proved to be a potent inhibitor of the rapid degradation of these mAb, by inhibiting internalization via an FcgammaRII-mediated mechanism. Both methods of reducing the degradation of radioiodinated mAb are expected to prolong irradiation of malignant b cells and consequently result in an enhanced therapeutic effect in vivo.

A new low-incidence antigen in the Kell blood group system: VLAN (KEL25).

A multilaboratory investigation has identified a new low-incidence antigen "VLAN' on the red cells of a blood donor. The VLAN antigen is destroyed by 2-aminoethylisothiouronium bromide treatment of the donor's red cells suggesting an association with the Kell system. Monoclonal antibody-specific immobilization of erythrocyte antigen analysis with anti-VLAN and with several mouse monoclonal antibodies directed at epitopes on the Kell glycoprotein gave positive results, indicating that the VLAN antigen is located on the Kell glycoprotein. The VLAN red blood cells have the common Kell phenotype: KEL:-1,2,-3,4,5,-6,7,-10,11,12,13,14,-17,18,19,-21,22,-23,-24. Additional serologic data indicate that the VLAN antigen is not part of any other ISBT blood group system, collection or series. A family study showed that the VLAN antigen is inherited since the red cells of two sisters and one niece of the propositus are also VLAN+. The ISBT Working Party on Terminology for Red Cell Surface Antigens has assigned VLAN to the Kell blood group system as KEL25 (number for computer listings 006025).

Prolonged persistence of PCR-detectable minimal residual disease after diagnosis or first relapse predicts poor outcome in childhood B-precursor acute lymphoblastic leukemia.

The follow up of minimal residual disease (MRD) in childhood B-precursor ALL by polymerase chain reaction (PCR) may be of help for further stratification of treatment protocols, to improve outcome. However, the clinical relevance of this approach has yet to be defined. We report the retrospective follow-up of MRD in bone marrow (BM) samples from 50 childhood B-precursor ALL patients by IgH/TCR delta PCR. Twenty-two patients remained in continuous complete remission (median follow-up 61 months), and 28 experienced relapse (median follow-up 75 months). Initial regression of MRD on therapy correlated with outcome. At the end of induction therapy 2/18 (11.1%) patients from the CCR group were PCR positive vs 10/16 (62.5%) from the 'relapse' group (P = 0.005). The presence of PCR detectable MRD predicted event-free survival independent of standard clinical and cytogenetical parameters. Also subsequent to first BM relapse, a correlation between MRD regression and outcome was observed. Six of eight patients who became PCR negative in the time period between relapse and bone marrow transplantation are in CCR, whereas 7/7 patients who remained PCR positive in this time period died (P = 0.006). In approximately 70% of evaluable patients, clinical relapse was preceded by recurrence of detectable MRD at time intervals of 3-18 months earlier and the recurrence of PCR positivity after a period of negativity was always followed by overt relapse. At relapse, the combined use of IgH and TCR delta probes reduced false negativity caused by clonal evolution to approximately 10%. This study shows that the evolution of PCR detectable MRD is an independent predictor of outcome.

Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia.

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti-Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse-transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA-3 allele.

Frequent ongoing T-cell receptor rearrangements in childhood B-precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease.

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.

Mitoxantrone is effective in treating childhood T-cell lymphoma/T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell malignancies in childhood are highly malignant diseases usually treated with intensive multidrug chemotherapy. In these regimens, anthracyclines, which are considerably cardiotoxic, are used. Mitoxantrone is structurally related to the anthracyclines, but it is considered to be less cardiotoxic. Therefore, the effectiveness of mitoxantrone in treating childhood T-cell malignancies was studied. METHODS: Fourteen newly diagnosed children with T-cell malignancies (12 T-cell non-Hodgkin's lymphoma, 2 T-cell acute lymphoblastic leukemia) initially were treated with one bolus injection of mitoxantrone, 12 mg/m2 intravenously, 1 week before standard induction therapy was begun. The effect of mitoxantrone was evaluated at Day 8, just before standard induction therapy was begun. RESULTS: Of 12 evaluable patients, 11 had significant positive responses with regard to the size of the primary tumor, the size of the involved peripheral lymph nodes, and the presence of lymphoblasts in the peripheral blood. The toxicity of mitoxantrone was mild. CONCLUSIONS: Mitoxantrone is effective in treating childhood T-cell malignancies. Its efficacy needs to be compared with the anthracyclines in a future randomized study.