Results of clot waveform analysis and thrombin generation test for a plasma-derived factor VIIa and X mixture (MC710) in haemophilia patients with inhibitors--phase I trial: 2nd report.

We reported the results of a clinical pharmacological study of MC710 (a mixture of plasma-derived FVIIa and FX) in haemophilia patients with inhibitors during a non-haemorrhagic state. This report provides the results of a clot waveform analysis (CWA) and thrombin generation test (TGT) using blood samples obtained in this study. CWA and TGT were conducted using blood samples obtained from a pharmacokinetic and pharmacodynamic study in which MC710 (five dose rates: 20, 40, 80, 100 and 120 µg kg(-1)) was compared with NovoSeven (120 µg kg(-1)) and FEIBA (two dose rates: 50 and 75 U kg(-1)) as control drugs in 11 haemophilia patients with inhibitors without haemorrhagic symptoms. CWA showed that MC710 provided significantly greater improvement than the control drugs in activated partial thromboplastin time (APTT) at 80 µg kg(-1); maximum clot velocity and maximum clot acceleration were more enhanced by MC710 than by control drugs. TGT revealed that MC710 significantly shortened the initiation time of thrombin generation in comparison to FEIBA and induced greater thrombin generation potency than NovoSeven. It was not clear whether or not MC710 caused significant dose-dependent changes in the two measurements; however, differences between MC710 and the control drugs were clarified. MC710 was confirmed to have superior coagulation activity and thrombin productivity and is expected to have superior bypassing activity.

Clinical pharmacological study of a plasma-derived factor VIIa and factor X mixture (MC710) in haemophilia patients with inhibitors--phase I trial.

MC710, a combined product of plasma-derived activated factor VII (FVIIa) and factor X (FX) at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In this study, pharmacokinetic (PK), pharmacodynamic (PD) parameters and safety of single doses of MC710 were investigated in 11 male haemophilia patients with inhibitors in a non-bleeding state. This was a multi-centre, open-labelled, non-randomized, active controlled crossover, dose-escalation study of five doses (20-120 µg kg(-1) of FVIIa) with re-administration of different MC710 dosages to the same subjects. The active controls were NovoSeven (120 µg kg(-1)) and/or FEIBA (50 and 75 U kg(-1)) which were used to compare PD parameters. The area under the curve (AUC) and maximum plasma concentration (C(max)) of MC710 active ingredients increased dose-dependently within the range of 20 and 120 µg kg(-1). After administration of MC710, activated partial thromboplastin time (APTT) was dose-dependently improved and prothrombin time (PT) was shortened to approximately 6 s at 10 min, and APTT improvement and PT shortening effects were maintained until 12 h after administration of MC710 at all doses. No serious or severe adverse event was observed after administration of MC710; furthermore, several diagnostic marker values and those changes did not indicate any signs of disseminated intravascular coagulation (DIC). These results suggest that MC710 would have haemostatic potential equal to or greater than NovoSeven and FEIBA and was be tolerable when given at doses up to 120 µg kg(-1).

An analysis of factors affecting the incidence of inhibitor formation in patients with congenital haemophilia in Japan.

Studies conducted in European and North American countries have demonstrated that various factors including races affect the frequency of inhibitor formation in haemophilia patients. The present study was undertaken to analyse factors affecting the incidence of inhibitor formation in Japanese haemophilia A and B patients. Analytical data were retrospectively collected from haemophilia A and B patients born after 1988, the year when monoclonal antibody-purified factor VIII products were first marketed in Japan. Various data were collected from 184 patients (153 cases of haemophilia A; 31 cases of haemophilia B). The sample size of haemophilia B cases was too small to reveal any significant differences between the inhibitor formation group and the inhibitor-free group in any of background variables. For patients with haemophilia A, on the other hand, univariate analysis identified the severity of haemophilia and a positive family history of inhibitor development as risk factors for the formation of inhibitors. In analyses of the clotting factor products used, the incidence of inhibitor formation did not differ significantly between the group treated with plasma-derived products (29.7%) and the group treated with recombinant products (25.0%). When background variables were compared, age was higher in the group treated with plasma-derived products but none of the other background variables differed between the two groups. These results suggest that in Japanese haemophilia patients, the type of clotting factor preparations used for therapy has not influenced the incidence of inhibitor formation.

Hepatoblastoma with congenital absence of the portal vein - a case report.

A 17-month-old girl who had been followed up as an extremely-low-birth-weight infant presented with hepatoblastoma in the right lobe of her liver. Preoperative angiography revealed an absence of the portal vein, and the visceral venous return was through the left renal vein into the inferior vena cava. No liver dysfunction and no jaundice were found; however, a marked elevation of the alpha-fetoprotein level was noted. She underwent a typical right hepatic lobectomy successfully after chemotherapy and has no evidence of recurrence 6 months after surgery.

Frequent aberration of FHIT gene expression in acute leukemias.

We analyzed the mRNA expression of the FHIT gene by reverse transcription-PCR (RT-PCR) in 54 cases of acute lymphoblastic leukemia (ALL; 11 cases of T-cell ALL [T-ALL] and 43 cases of non-T-ALL) and 40 cases of acute myeloid leukemia (AML). In 46% of the ALL cases and 55% of the AML cases, FHIT expression was absent or markedly decreased. Only abnormal short bands were detected in 30% of the ALL cases and 5% of the AML cases. Eighteen of 19 abnormal transcripts had the same fusion of exons 2-7, and all lacked the starting codon in exon 5. No obvious normal-sized PCR products were detected in cases exhibiting abnormal transcripts. These findings suggest that the expression of functional FHIT protein was lost in the majority of ALL (76%) and AML (60%) cases. Differential quantitative PCR of exons 3-9 of the FHIT gene and RT-PCR of the PTPRG gene, which is centromeric to the FHIT gene, showed the presence of the target sequences. Fluorescence in situ hybridization analysis using probes covering exons 5 and 8 revealed no difference in the signal patterns between leukemia and normal cells, showing one or two signal doublets in more than 90% of nuclei, and indicated that gross segments of the FHIT gene were not homozygously deleted in these cases. A small number of transcripts with an aberrant fusion between exons 2 and 7 were detected by RT-PCR in the bone marrow cells from four healthy individuals. Granulocytes, lymphocytes, and monocytes in the bone marrow cells of a healthy individual contained transcripts with the same fusion. This unique fusion of exons 2 and 7 might be preferentially seen in either neoplastic or normal hematopoietic cells, regardless of their lineage. The finding that FHIT expression was abolished in the majority of leukemia cases might support the hypothesis that the FHIT gene acts as a tumor suppressor, at least in leukemia.

Long-term survivors of advanced neuroblastoma with MYCN amplification: A report of 19 patients surviving disease-free for more than 66 months.

PURPOSE: According to initial reports, stage 4 neuroblastoma patients with amplification of the MYCN proto-oncogene developed progressive disease within 8 months. The prognosis for such patients, however, should now be reevaluated in light of recent results achieved with up-to-date combination chemotherapy. PATIENTS AND METHODS: Patients with stage 3, 4, and 4S neuroblastoma and more than 10 copies of MYCN received induction chemotherapy, which from January 1985 to February 1991 consisted of regimen A(1 )(cyclophosphamide 1,200 mg/m(2) on day 1, vincristine 1.5 mg/m(2) on day 1, pirarubicin 40 mg/m(2) on day 3, and cisplatin 90 mg/m(2) on day 5) and from March 1991 to September 1993 consisted of regimen A(3 )(cyclophosphamide 1,200 mg/m(2) on days 1 and 2, pirarubicin 40 mg/m(2) on day 3, etoposide 100 mg/m(2) on days 1 through 5, and continuous infusion cisplatin 25 mg/m(2) on days 1 through 5). Most of these patients underwent radical surgery to remove the original tumor and local metastases, irradiation, and supralethal preconditioning regimens, followed by blood stem-cell transplantation (SCT). Data on the patients were collected in December 1998, and the factors contributing to disease-free survival were analyzed. RESULTS: During the study period, 66 patients with more than 10 copies of MYCN were treated. Five of nine patients with stage 3 disease, 13 of 55 with stage 4, and one of two with stage 4S survived for at least 66 months. It is interesting that all but one patient who survived for more than 66 months underwent SCT, in contrast with only five of 45 patients who died. CONCLUSION: Not all patients with advanced neuroblastoma who have more than 10 copies of MYCN will die. The requisites for survival in such patients seem to be intensive induction chemotherapy, effective surgery, irradiation, and the use of SCT.

Clinical characteristics of infant acute leukemia with or without 11q23 translocations.

Of 34 infants less than 1 year of age with acute leukemia, 20 had an 11q23 translocation (group I), 8 had t(4;11), 5 had t(11;19), 3 had t(1;11), 2 had t(10;11), 1 had t(9;11), and the other had an 11q+ chromosome. Nine had other chromosome changes (group II), including t(1;19), t(8;14), 5q- chromosome, or +8 in one each, and a translocation involving 7p22 in two. The other five had normal diploidy in their leukemic cells (group III). Thus, the 11q23 translocation was seen in 50% of the leukemic infants, and in as high as 75% of the infants less than 6 months old. While the 7p22 translocations were both seen in those less than 6 months, the four chromosome abnormalities without 11q23 translocation mentioned above and normal diploidy were found only in those 6 months old or more. The group I patients had higher leukocyte counts than the group II (p less than 0.05) or group III (p less than 0.01) patients. Of the 20 group I patients, 16 were classified as having ALL, and 4 were classified as having ANLL. Eleven of 15 ALLs with the 11q23 translocation showed an Ia+, CALLA-, and B4+ (8 of 9 examined) immunophenotype. Coexpression of lymphoid and myeloid Ags was seen in four ALLs and two ANLLs with the 11q23 translocation. The survival of group II patients (median, 9 months) was significantly shorter than that of group I (median, 19 months) (p less than 0.05) or group III (median, 44 months) (p less than 0.01) patients; the difference in the survival between group I and group III patients was not significant. It is noteworthy that 5 of the 20 group I patients have survived 20 months or more without relapsing.

Internal tandem duplication of the FLT3 gene and clinical evaluation in childhood acute myeloid leukemia. The Children's Cancer and Leukemia Study Group, Japan.

We analyzed tandem duplication in the juxtamembrane (JM) domain of the FLT3 (FMS-like tyrosine kinase 3/FLK2, CD135) gene in 94 children with acute myeloid leukemia (AML) and evaluated its correlation with clinical features. Longer polymerase chain reaction (PCR) products were observed in five patients; 1/3 of M0, 119 of M1, 1/39 of M2, 1/9 of M3 and 1/12 of M5. The sequence analyses of abnormal PCR products showed that all the abnormal products were derived from tandem duplications involving the JM domain and that all the lengthened sequences were in-frame as we previously reported. Statistical analyses revealed a significantly lower incidence of the tandem duplication in childhood AML patients than in adult patients (P < 0.05), and significantly shorter disease-free survival in patients with mutant FLT3 than in patients with wild-type FLT3 (P < 0.05). Our results suggest that the tandem duplication in the JM domain of the FLT3 gene is not a frequent phenomenon but might be a factor of poor prognosis in childhood patients with AML.

Polymorphism of CCR5 affecting HIV disease progression in the Japanese population.

Among several factors associated with HIV-1 disease progression, genetic polymorphism of CCR2, CCR5, and CXCR4 in HIV-1 infection has been found. Single-nucleotide polymorphisms (SNPs) in the CCR2, CCR5, and CXCR4 genes as well as a 32-base pair deletion in the open reading frame of the CCR5 gene are associated with HIV disease progression among Caucasians and African-Americans in North America and Europe. However, in populations other than Caucasians and African-Americans, SNPs have not been fully examined. In our study SNPs in CCR2 coding and CCR5 regulatory regions have been examined in 98 Japanese HIV-positive individuals. The alleles of CCR5 regulatory regions at -2135T and -2086G are associated with late onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 0.502 and 0.404, respectively). In contrast to this, the allele of CCR5 at -2086A is associated with the early onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 2.133). A haplotype including two alleles at -2135G and -2086G is associated with the late onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 0.372). Thus we found that a CCR5 SNP and haplotype polymorphism affect HIV disease progression even in the Japanese population. This indicates that the CCR5 genetic polymorphism affecting disease progression should be studied in a wider range of population.

Correlation of titer of antibody to principal neutralizing domain of HIVMN strain with disease progression in Japanese hemophiliacs seropositive for HIV type 1.

With the use of the principal neutralizing determinant (PND) peptide-based ELISA to measure anti-PND antibodies that specifically bound synthetic peptides derived from HIVIIIB, HIVMN, HIVRF, HIVSC, HIVWJM-2, HIVAf1l.con, or HIVAf2.con, type-specific antibodies to the HIVMN peptide were studied in 350 serum specimens from Japanese with hemophilia A who had been injected with known unheated factor VIII concentrates until 1985 and had been infected with HIV-1 subtype B. These antibodies were not found in any of the seronegative sera of hemophiliacs, patients with autoimmune diseases, or normal healthy controls. Further, all hemophiliacs rapidly progressing to AIDS and death among the 95 hemophiliacs in a restricted Nara area had antibody titers of less than 20 and their low levels preceded the rapid progression to the disease state. In contrast, slowly progressing hemophiliacs maintained an antibody titer of more than 100 from the initial stages of viral infection and remained asymptomatic. Sequence analysis of the V3 regions of HIV-1 indicated that the hemophiliacs who maintained a high anti-PNDMN antibody level showed a conserved MN sequence. In contrast, the HIV-infected hemophiliacs with nonreactivity in the ELISA showed sequence changes in the neutralizing epitopes of HIVMN. The dynamic of the serum anti-PNDMN antibody titer appear to be a characteristic indicator of the progression of the HIV-infected status in Japanese hemophiliacs seropositive for HIV-1.

Effects of granisetron in children undergoing high-dose chemotherapy: a multi-institutional, cross-over study.

The efficacy of granisetron hydrochloride 20 microg/kg and 40 microg/kg were compared using a cross-over method to determine the optimal dose in children with solid tumors receiving high-dose chemotherapy. Granisetron controlled the onset of vomiting in 17 of 23 patients (73.9%) who were given 40 microg/kg of granisetron, while 8 of 21 patients (38.1%) were free of vomiting in the 20 microg/kg group. The average frequency of vomiting was 7.22 times in the 20 microg/kg dose versus 4.44 times in the 40 microg/kg dose. No safety problems were associated with either dose. The 40 microg/kg dose of granisetron appears to be more optimal.

Time-course detection of HIV-1 proviral DNA and genomic RNA by polymerase chain reaction in sera from seropositive and seronegative hemophiliacs treated with clotting factor concentrates.

The detection of HIV-1 proviral DNA and genomic RNA was performed by polymerase chain reaction (PCR) in hemophiliacs treated with non-heated clotting factor concentrates. Reamplification with double PCR was performed on those samples that were negative for single PCR. Primer pairs of the gag, env, and pol regions were used for the amplification of HIV-1 proviral DNA sequences. Amplification of the gag region by the SK38/SK39 primer pair was useful for the detection of proviral DNA sequences. With double PCR, 44 of 47 seropositive samples (93.6%) were PCR-positive. All 23 seronegative samples were PCR-negative. Reverse transcription and PCR amplification (RT-PCR) according to the primer pair of the gag region were performed to detect HIV-1 genomic RNA sequences. Double RT-PCR analysis of the HIV-1 RNA sequence in frozen-preserved sera revealed that 49 of 55 seropositive sera (89.1%) were PCR-positive. Although quantification of the PCR method was not performed in this study, we concluded that, in patients in whom proviral DNA or genomic RNA sequences are detected with difficulty with PCR, the onset and progression of HIV-1 infection is delayed.

Treatment outcome and prognostic factors in childhood acute myeloblastic leukemia: a report from the Japanese Children's Cancer and Leukemia Study Group (CCLSG)

Treatment outcome and prognostic factors were evaluated in 152 children with acute myeloblastic leukemia (AML) treated on three consecutive protocols (ANLL 861, 8912, 9205) of the Children's Cancer Leukemia Study Group (CCLSG, Japan). In the ANLL 9205 protocol, anthracycline was used with a continuous infusion of cytosine arabinoside, followed by an intensive sequential post remission chemotherapy of short duration. Forty-two of these 46 patients (91.3%) achieved complete remission, and 58.8% of these patients projected a 3-year disease-free survival. These results were apparently superior to those obtained with the ANLL 861 and 8912 protocols, which used conventional doses of multiple drugs followed by a moderate post remission chemotherapy of long duration. This favorable response with the ANLL 9205 protocol was attributed mainly to the high induction rate of patients with the M4 and M5 FAB subtypes, as compared to those in the previous two protocols (93.3% in ANLL 9205 vs. 57.9% in ANLL 861 + 8912; P < 0.05). The ANLL 861 and 8912 protocols, an older age (> or = 8 years), higher WBC counts (> or = 10 x 10(9)/1) and all predicted an increased risk of relapse and decreased the survival following univariate analysis (P < 0.05). An older age and high WBC count continued to predict an increased risk of relapse in multivariate analyses: patients with an age > 8 years and WBC counts > 10 x 10(9)/1 had a 4.5 times higher risk of relapse than patients without these adverse features.

Retrospective analysis of late intensification therapy with high-dose methotrexate for standard-risk acute lymphoblastic leukemia in childhood (CCLSG-S811 study). The Children's Cancer and Leukemia Study Group.

Using the CCLSG-S811 protocol for children with standard-risk acute lymphoblastic leukemia (ALL), late intensification therapy (LIT) with high-dose methotrexate (HD-MTX) was conducted without randomization. Of 118 eligible patients, 114 attained complete remission and 82 maintained continuous complete remission (CCR) for at least 3 years, completing the entire S811 regimen. Among the latter, 74 patients received LIT with HD-MTX between 2-3 years after CCR onset. MTX (2,000 mg/m2 per dose per week) was administered by 24 h infusion and three doses were given every 12 weeks. Leucovorin rescue (15 mg/m2 i.v.) every 6 h was initiated 12 h after the end of MTX infusion for seven doses. As regular maintenance chemotherapy, intermittent (Regimen A) or continuous (Regimen B) MTX plus 6-mercaptopurine (6MP) combined with pulses of prednisolone and vincristine was administered (Koizumi S, Fujimoto T, Takeda T, et al. Cancer 1988; 61: 1292-1300). Retrospective analysis revealed that patients on Regimen A who started LIT earlier (within 2 years of CCR onset (n = 23)) showed a higher rate of event-free survival (EFS) at 8 years (95.5% +/- 4.4%, mean +/- S.E.) than patients who started LIT later (2.5 years after CCR onset (n = 18, 66.2% +/- 11.3%, p less than 0.01)). In addition, the superiority of four or five courses of the LIT (n = 39) as compared to 2 or 3 courses (n = 35) was noted for both regimens. The data suggest that early and aggressive LIT with HD-MTX may improve the long-term survival of childhood ALL patients.

Mutational analysis of the N-ras gene in acute lymphoblastic leukemia: a study of 125 Japanese pediatric cases.

A point mutation of the N-ras gene is one of the known genetic alterations identified in patients with acute lymphoblastic leukemia (ALL), but its clinical importance is still controversial. Using polymerase chain reactions, we examined codons 12, 13 and 61 of this gene in 125 Japanese childhood ALL patients (64 common-ALL, 22 pre-B-ALL, 33 T-ALL, 2 B-ALL, 3 undifferentiated ALL, and 1 unclassified ALL) including 9 relapsed patients. An N-ras point mutation was observed in 14 (11%) patients (9 common-ALL, 3 T-ALL, and 2 undifferentiated ALL; 13 patients at diagnosis and 1 at relapse). The patients with undifferentiated ALL harbored an N-ras mutation at a significantly higher rate. However, no correlation was found between the presence of an N-ras mutation and sex, age, or white blood count. There was no significant difference in the event-free survival rate between 13 fresh patients with an N-ras mutation and 103 patients with a wild-type configuration. The N-ras mutation was present in about 10% of childhood ALL cases but it did not have a prognostic impact. The sequence analyses revealed that the majority of the patients (13/14) had an N-ras mutation of a G to A transition. This finding was consistent with previous reports on N-ras mutations in acute leukemias in which the incidence of a G to A mutation was significantly higher in ALL than in myeloid malignancies.

Prognostic impact of CD45 antigen expression in high-risk, childhood B-cell precursor acute lymphoblastic leukemia.

To evaluate the clinical implications of CD45 expression in acute childhood lymphoblastic leukemia (ALL), we measured the CD45 expression of blast cells from 133 untreated patients with childhood B-precursor ALL (n = 118) or T-ALL (n = 15). CD45 expression (> or = 20%) was detected in all 15 cases (100%) of T-ALL, and 101 cases (86%) of B-precursor ALL. In 122 cases, the fluorescence intensity of the CD45 expression was measured as a relative value; the ratio of average linear values (RALV) of CD45 on the blasts to that on CD3-positive T-lymphocytes from the same specimen. The expression was more intense in the T-ALL cases than in the B-precursor ALL cases (RALV, mean +/- SE: T-ALL 0.230 +/- 0.04 vs. pro-B ALL 0.150 +/- 0.012/pre-B ALL 0.153 +/- 0.019, p < 0.05). However, the intensity of the CD10, CD19, CD20 and CD34 antigen immunoreactivity did not correlate with the CD45 expression. Patients with hyperdiploidy (chromosome number > 50) showed significantly lower levels of CD45 expression than patients with t(1;19) or normal karyotypes (RALV, mean +/- SE: 0.081 +/- 0.022 vs. 0.133 +/- 0.03/0.143 +/- 0.019, p < 0.05). Other clinical features such as age, gender and WBC count did not correlate with CD45 expression. The prognostic implications of CD45 expression were studied in non-high-risk (low-risk + intermediate-risk) (n = 60) and high-risk patients (n = 52) with B-precursor ALL who had been treated with the risk-directed protocol of ALL-941 trial. Although CD45 expression did not correlate with the event-free survival (EFS) of the non-high-risk patients, there was a significant correlation between the expression levels and the EFS of the high-risk patients: the 3-year EFS rate of the CD45low group (n = 26, RALV = 0.017-0.132) was 88 +/- 7% versus the CD45high group (n = 26, RALV = 0.133-0.450) at 34 +/- 24% (p < 0.05). These results show that the levels of expression of the CD45 antigen on leukemic lymphoblasts are significantly correlated with the clinical features and prognosis of childhood ALL.

[HIV infection: pathophysiology and its clinical features--clinical course and surrogate marker].

Clinical courses and several hematological and serological markers were evaluated in 34 patients with HIV infection (32 with hemophilia and 2 with homosexuality) who had been followed up at Shizuoka Prefecture Children's Hospital for 3 to 13 years, with the reference to the results observed in 1,082 patients with HIV infection registered with the Ministry of Health and Welfare Study Group, Natural History Committee. The results indicated as significant follow-up markers as (1) CD4- and CD8-positive lymphocyte count, (2) IgA, IgG and beta-2 microglobulin, (3) HIV p24 antigen and HIV culture isolation, and (4) serum HIV-RNA. Of these markers, the quantification of serum HIV-RNA seemed particularly useful for prognosis estimation, determination of therapy onset, and efficacy assessment of a given anti-HIV agent. In addition, we encountered 2 HIV-infected patients who seemed to meet the definition of long-term survivors. Factor analysis in these patients will be an important subject of investigation from a clinical point of view as well.

[Hemopoietic factor producing hepatocellular carcinoma in a child].

A 12-year-old boy with hepatocellular carcinoma presented with marked neutrophilia up to 69,200/microliters and elevation of serum colony stimulating activity (CSA) and burst promoting activity (BPA). After resection of the tumor, neutrophil count returned to the normal value and hemopoietic activities in serum decreased. However, on relapse, both elevated to the preoperative levels. Hemopoietic activities in the medium conditioned with tumor tissue were investigated using normal human bone marrow as the target cells, and both CSA and BPA were demonstrated. From these findings, it could be considered that the tumor tissue produced a substance that stimulated both myelopoiesis and erythropoiesis.

[Detection of HIV-1 proviral DNA and RNA in serum from HIV-1 sero-positive hemophiliacs by polymerase chain reaction].

We amplified HIV-1 proviral DNA and RNA in serum by DNA-PCR and RT-RCR in 20 HIV-1 seropositive hemophiliacs asymptomatic carrier (AC): 10, AIDS-related complex (ARC):8, AIDS:2). HIV-1 DNA sequences were identified in 44 (93.6%) of 47 specimens. 23 (92.0%) of 25 samples in AC, 13 (100%) of 13 in ARC, 8 (88.8%) of 9 in AIDS had detectable bands. RNA sequences were identified in 36 (85.7%) of 42 specimens. 17 (70.8%) of 24 specimens in AC, 13 (100%) of 13 in ARC, 5 (100%) of 5 in AIDS cases had detectable bands. RNA in serum was strongly positive in two asymptomatic patients with progression to AIDS who showed no changes of other markers. Patients with almost normal CD4 cell counts had trace amounts of proviral DNA and RNA in serum. RNA in serum is the parameter most predictive of progression to AIDS and is detectable earlier than other parameters. From the results of RT-PCR before and after seroconversion, the serum of one patient was shown to be positive at 5 months before seroconversion.

[Analysis of cytoplasmic antigens in acute leukemia by flow cytometry].

The expression of cytoplasmic antigens in 77 cases of acute leukemia were analyzed by flow cytometry using the following monoclonal antibodies: CD3, CD22, anti-myeloperoxidase (MPO-7) and anti-mu-heavy chain. CD22 antigen was detected in the cytoplasm of all non-T-ALL patients excluding one not-tested patient. In two patients with unclassified ALL, surface CD22 antigen was not expressed but cytoplasmic CD22 antigen was strongly expressed. Three out of 9 patients with common ALL were cytoplasmic mu-heavy chain-positive, so these patients were diagnosed as Pre-B ALL. In four out of 8 patients with T-ALL, CD3 antigen was not expressed on the cell surface membrane. However all of T-ALL patients excluding one non-tested patient were cytoplasmic CD3-positive. The cytoplasmic expression of myeloperoxidase antigen was detected in twenty out of 21 patients with acute non-lymphoblastic leukemia (ANLL). One megakaryocytic leukemia patient was MPO-negative. In two ANLL patients, the percentage of MPO for conventional cytochemical staining was undetectable or low, but MPO antigens were positive (77% and 70%) for flow cytometric analysis. All of 46 non-T ALL patients were cytoplasmic MPO-negative, however 4 out of 10 T-ALL patients were cytoplasmic MPO-positive. The study proved that the analysis of cytoplasmic CD3, CD22, mu-chain and MPO antigens were very useful to define the cell lineage of leukemia and to classify ALL and ANLL. It is necessary to study further whether the expression of MPO in the cytoplasm of T-ALL was non-specific reaction or whether MPO precursors are expressed in the cytoplasm of T-ALL.