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.

Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase.

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.

Marginal benefit/disadvantage of granulocyte colony-stimulating factor therapy after autologous blood stem cell transplantation in children: results of a prospective randomized trial. The Japanese Cooperative Study Group of PBSCT.

In this prospective trial, a total of 74 children who were scheduled to undergo high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (PBSCT) were prospectively randomized at diagnosis to evaluate the effectiveness of exogenous granulocyte colony-stimulating factor (G-CSF) treatment in accelerating hematopoietic recovery after PBSCT. The diagnosis included acute lymphoblastic leukemia (ALL) (n = 27), neuroblastoma (n = 29), and miscellaneous solid tumors (n = 18). Eligibility criteria included (1) primary PBSCT, (2) chemotherapy-responsive disease, and (3) collected cell number >1 x 10(5) colony-forming unit-granulocyte-macrophage (CFU-GM)/kg and >1 x 10(6) CD34(+) cells/kg patient's body weight. After applying the above criteria, 11 patients were excluded due to disease progression before PBSCT (n = 6) or a low number of harvested cells (n = 5), leaving 63 patients for analysis; 32 patients in the treatment group (300 microg/m2 of G-CSF intravenously over 1 hour from day 1 of PBSCT) and 31 in the control group without treatment. Two distinct disease-oriented high-dose regimens without total body irradiation consisted of the MCVAC regimen using ranimustine (MCNU, 450 mg/m2), cytosine arabinoside (16 g/m2), etoposide (1.6 g/m2), and cyclophosphamide (100 mg/kg) for patients with ALL, and the Hi-MEC regimen using melphalan (180 mg/m2), etoposide (1.6 g/m2), and carboplatinum (1.6 g/m2) for those with solid tumors. Five patients (two in the treatment group and three in the control group) were subsequently removed due to protocol violations. All patients survived PBSCT. The median numbers of transfused mononuclear cells (MNC), CD34(+) cells, and CFU-GM were, respectively, 4.5 (range, 1 to 19) x 10(8)/kg, 8.0 (1.1 to 25) x 10(6)/kg, and 3.7 (1.2 to 23) x 10(5)/kg in the treatment group (n = 30) and 2.9 (0.8 to 21) x 10(8)/kg, 6.3 (1.1 to 34) x 10(6)/kg, and 5.5 (1.3 to 37) x 10(5)/kg, respectively, in the control group (n = 28), with no significant difference. After PBSCT, the time to achieve an absolute neutrophil count (ANC) of >0.5 x 10(9)/L in the treatment group was less than that in the control group (median, 11 v 12 days; the log-rank test, P =.046), although the last day of red blood cell (RBC) transfusion (day 11 v day 10) and the duration of febrile days (>38 degrees C) after PBSCT (4 v 4 days) were identical in both groups. However, platelet recovery to >20 x 10(9)/L was significantly longer in treatment group than control group (26 v 16 days; P =.009) and >50 x 10(9)/L tended to take longer in the treatment group (29 v 26 days; P =.126), with significantly more platelet transfusion-dependent days (27 v 13 days; t-test, P =.037). When patients were divided into two different disease cohorts, ALL patients showed no difference in engraftment kinetics between the G-CSF treatment and control groups, while differences were seen in those with solid tumors. We concluded that the marginal clinical benefit of 1 day earlier recovery of granulocytes could be offset by the delayed recovery of platelets. We recommend that the routine application of costly G-CSF therapy in children undergoing PBSCT should be seriously reconsidered.

[Bone marrow relapse in high-risk pediatric patients with acute lymphoblastic leukemia: a comparison of relapse times and initial clinical features of patients on different protocols. Children's Cancer and Leukemia Study group (CCLSG)].

To clarify the efficacy of modern intensive chemotherapy for ALL patients with unfavorable features, we compared the time to failure and initial clinical features of children who relapsed in the bone marrow or combined sites, as documented by early CCLSG studies (H811 and H851; 1981-1987) and later studies (H874 and H/HH911; 1987-1993) concerning high-risk ALL patients. In the later studies patients outcomes with new intensive regimens employing early intensification and reinduction therapy were apparently better than those of patients in the early studies with conventional regimens. When we compared the number of relapsed patients based on duration of first remission, we found that the improved outcomes for patients in the later studies were due to a decrease in the number who relapsed 7-36 months after the start of treatment (intermediate relapse), and that the percentage of those who relapsed within the first 6 months of therapy (early relapse) was higher. Patients with high initial WBC counts tended to relapse much earlier than those with low initial WBC counts. However, in the later studies, patients with high WBC counts often relapsed after the termination of therapy (late relapse). These results suggest that the intensive chemotherapy regimens used in the later studies can prevent the development of drug resistant leukemic clones, except in extremely high-risk patients likely to relapse within the first 6 months of therapy.

Long-term effects of bone marrow transplantation for inborn errors of metabolism: a study of four patients with lysosomal storage diseases.

Long-term effects of bone marrow transplantation (BMT) were evaluated in patients with I-cell disease, metachromatic leukodystrophy (MLD), Maroteaux-Lamy syndrome or Hunter syndrome (mild form). Donors were human leukocyte antigen (HLA)-matched siblings, and the follow-up periods were 24-71 months after BMT. The enzyme activities were increased in leukocytes, plasma or liver tissues compared with pre-BMT levels. A patient with I-cell disease acquired development of 4-8 month old infants and showed no further progression in cardiac dysfunctions. A patient with MLD showed a decelerated disease progression and an improved peripheral neuropathy, but progressive brain atrophy was not prevented. Patients with Maroteaux-Lamy syndrome or Hunter syndrome showed improvements in hepatomegaly, joint contractures, short stature and tight skin, and this greatly increased their quality of life. These results indicated that the long-term therapeutic effects achieved by BMT were subject to multiple factors including biochemical improvements, a reversibility of affected tissues, or advanced states of disease and central nervous system impairments in inborn errors of metabolism.

A sensitive colorimetric detection of virus DNA and oncogene.

Advantage of cloning probe DNA fragment in phage M13 DNA was taken to provide a larger single stranded DNA as a hybridization probe. High level of direct enzyme labels was introduced via the M13 DNA moiety as well as probe DNA. A highly sensitive colorimetric detection of virus DNA and oncogene was developed.