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To investigate the efficacy of the combination of ultrasound-guided rectus sheath (RS) and transversus abdominis plane (TAP) blocks compared with TAP or RS block alone in gynecological single-incision laparoscopic surgery (SILS).Bilateral TAP blocks (Group A, n = 12), TAP and RS blocks (Group B, n = 12), and RS blocks (Group C, n = 12) with 40 ml ropivacaine/patient were performed for ovarian tumor SILS. The analgesic effects were evaluated using a numerical rating scale (NRS) at zero, six, 12, 24, and 48 hours post-surgery.Umbilical pain on completion of general anesthesia was significantly less frequent in Group B (1/12) than Group A (7/12) (p = 0.03). The postoperative NRS scores were significantly lower in Group B than Group A at zero (p = 0.02) and six (p = 0.03) hours and Group C at zero (p = 0.001), six (p = 0.02), and 12 (p = 0.004) hours.The combination of RS and TAP blocks reduced early postoperative pain compared with RS or TAP block alone for gynecological SILS.

To evaluate the effectiveness of ultrasound-guided transversus abdominis plane (TAP) and rectus sheath (RS) blocks in pain management and recovery after gynecological single-incision laparoscopic surgery (SILS).Abilateral TAP block (Group A, n = 9), bilateral TAP and RS blocks (Group B, n = 10), and a bilateral RS block (Group C, n = 9) with 40 ml ropivacaine per patient were conducted in 28 patients undergoing SILS for ovarian tumors. A pain score and walking distance in a 6-minute walk test (6MWT) were examined.Pain scores were significantly lower on postoperative day (POD) 3 than on POD 1 in Groups B (p = 0.03) and C (p = 0.02). The walking distance on POD 3 was comparable with that before surgery in Group C (p = 0.75), but shorter in Groups A (p = 0.004) and B (p = 0.02).The RS block alone was the most effective in relieving pain and accelerating general recovery after gynecological SILS.

We have simultaneously measured the field dependence of transverse voltages at two pairs of transverse probes along with the magnetoresistance to detect the motion of ferromagnetic domain walls in a thin NiFe wire. We observed sharp steplike changes of the transverse voltage correlated with the jumps in magnetoresistance, which have been reported previously in thin ferromagnetic wires. The field dependence is explained in terms of the rotation of local magnetization near the transverse probes. This fact suggests that such a local magnetization rotation might contribute to some of the negative domain-wall resistivity reported in recent papers.

(2000). Hb I-High Wycombe [β59(E3)Lys→Glu]: The First Instance in Japan. Hemoglobin: Vol. 24, No. 2, pp. 153-156.

A metallic Fe specimen, implanted with 111In, was oxidized and subsequently annealed in a high vacuum for PAC spectroscopy. This treatment gave rise to a huge PAC signal. The magnitude of the hyperfine field was found to be one third of that in metallic Fe. CEMS on an enriched Fe foil given exactly the same treatment has revealed that a maghemite phase (γ-Fe2O3) is formed right after the oxidation treatment and a magnetite phase (Fe3O4) after the vacuum annealing. 111In in the magnetite phase was found to give rise to a PAC signal with large amplitude. PAC spectroscopy in an external magnetic field has revealed that the site of 111In is the tetrahedral site of the magnetite with the hyperfine field of +12 T, which is in excellent agreement with those in the ferrites. The present method of oxidation of metallic Fe with nuclear probes in it is quite useful for the study of oxidation processes. Also, it provides us with a simple means to prepare ferrite specimens incorporated with nuclear probes.

Time-dependent analysis of rubber flow from a capillary rheometer is analyzed by the finite element method, in which both marker-particle and pseudo-concentration methods are used to define extruding rubber positions. The rubbers are assumed to be represented by a power-law fluid. The extruded free surface obtained by the marker-particle method is compared with the one produced by the pseudo-concentration method.

p16 and p15 genes are putative tumor suppressor genes located on chromosome 9p21. In acute leukemias, alterations of p16 and p15 genes have been reported to occur exclusively in lymphoid lineage. We analyzed alterations of p16 and p15 genes in 46 acute leukemias with MLL gene rearrangements by Southern blot analysis, and investigated the association with clinical characteristics. We identified homozygous deletion of p16 and p15 genes in five (19%) of 27 acute lymphoblastic leukemias (ALLs) and in two (11%) of 19 acute myeloid leukemias (AMLs). Patients with homozygous deletion of p16 and p15 genes showed higher average leukocyte counts (343 x 10(9)/l vs 271 x 10(9)/l) and lower estimated 2-year survival rates than those with normal p16 and p15 genes (14.3 vs 30.7%), although the differences were not statistically significant. In addition, we investigated mutation of p16 gene by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 31 patients, but no mutation was found in the patients tested. Our results suggest that alterations of p16 and p15 genes are involved in a subset of acute leukemias with MLL gene rearrangement not only of lymphoid but also of myeloid phenotype. Homozygous deletion of p16 and p15 genes may be a possible adverse prognostic factor, although further analysis would be needed to confirm it.

The clinical course of 48 children with chronic hepatitis C (33 boys, 15 girls; mean age: 12.2 years) was monitored for more than 3 years to clarify its natural course. All patients were positive for the second-generation antibody to hepatitis C virus (anti-HCV) and for serum hepatitis C virus (HCV) RNA. All but one patient had a history of blood transfusion. Serum levels of alanine aminotransferase (ALT) had been abnormal for more than 1.5 years. Spontaneous remission defined as a biochemical remission lasting more than 1 year in association with the disappearance of serum HCV RNA, occurred in 4 (8.3%), however, in 25%, HCV RNA was still detectable in the liver even after its disappearance from serum. In this patient, the level of antibody to HCV core antigen (anti-HCV core) did not decrease significantly and serum HCV RNA eventually reappeared. The serum titre of HCV RNA in the 4 children with spontaneous remission was lower than in the remaining 44 children. Spontaneous remission may occur in children with chronic hepatitis C in whom the serum titre of HCV RNA is low and serum level of anti-HCV core decreases significantly. Assessment of the intrahepatic HCV RNA is necessary to confirm complete remission. Conclusion A low serum titre of HCV RNA and a significant decrease in the serum titre of anti-HCV core were associated with spontaneous remission in children with chronic hepatitis C. Intrahepatic HCV RNA assessment is necessary to confirm complete remission.

Die swells of fluid flowing through the Monsanto Processability Tester (MPT) are calculated, A finite element method using six-node triangular isoparametric elements is used, to analyze the transient flow motion in which marker particles are introduced to represent the motion. Marker positions are determined by the use of a triangular area co-ordinate system Three simple examples of extruding processes for Newtonian fluid flows are described.

Fe/M (M = Ag, Zn and Sn) multilayers prepared by a vacuum evaporation method are studied by Mossbauer spectroscopy (MS), Rutherford backscattering spectroscopy (RBS) and X-ray diffraction (XRD). For the case of an M = Ag multilayer, MS reveals that Fe in the multilayer remains as anα-phase down to the layer thickness of 10 nm. This result is in agreement with the RBS result that Fe and Ag form a completely discrete layer structure without any mutual mixing. For the case of M = Zn and Sn, RBS reveals that a considerable mixing has taken place between Fe and Sn during the specimen preparation. MS on Fe/Sn specimens with different layer thickness shows that an alloy phase of about 5 nm thickness is formed at the interface. Structural as well as magnetic properties of the alloy phase are discussed based on MS at different temperatures and on reported results of the intermetallic compound FeSn.

Allogeneic bone marrow transplantation (BMT) in a patient with myelodysplastic syndrome (MDS) infected by hepatitis B virus (HBV) precore mutant: favorable outcome with slow tapering of immunosuppressive agents

We report a pediatric case of CAEBV and T cell-based Hodgkin's-like disease successfully treated with allo PBSCT from an HLA-matched sibling. The diagnosis of CAEBV was made from clinical signs and the presence of the EBV genome in PBMC and tumor cells. Conditioning with busulfan (BU) + etoposide (VP16) + cyclophosphamide (CY) was effective and well tolerated. EBV was totally eradicated by 3 months after allo PBSCT. Although she suffered from chronic GVHD of the liver, she has been well and free of disease for 47 months since PBSCT. We suggest allo PBSCT for CAEBV as a potent therapeutic strategy for eradication of the EBV genome and allowing immunological reconstitution.

The clonal composition of EBV-infected cells was examined in three cases of EBV-associated hemophagocytic syndrome by analysis of the heterogeneity of terminal repetitive sequences in the EBV genome, indicating monoclonal expansion of EBV-infected cells in all cases. Involvement of T lymphoid cells was determined by in situ hybridization using 35S-labeled RNA probes specific for the small EBV-encoded nuclear RNAs, EBER1 and EBER2, in combination with immunostaining for the TCR-beta chain, CD45RO, CD20, CD30 and CD68 antigens in these three cases. The majority of lymphoid cells showing EBER transcripts were stained by antibodies against CD45RO and T cell receptor-beta. In contrast, EBER-specific signals were not detectable on B cells or hemophagocytic cells. These data support the concept that EBV-associated T cell proliferation is a primary feature of EBV-AHS.

Fe/M (M = Ag, Sn) multilayer films have been prepared by a vacuum evaporation method. RBS is performed to examine the depth profile of the multilayer films. For M = Ag, a layer structure with a well defined interface is formed by the present evaporation method. Mossbauer spectroscopy as well as XRD shows that the Fe in the multilayer is in an α-Fe state. For M = Sn, RBS showed that a considerable mixing had taken place between Fe and Sn during the multilayer procedure.

The time differential perturbed angular correlation (PAC) method with111In(111Cd) as the probe was applied to study diffusion processes in cadmium as well as in dilute cadmium-indium alloys between 300 K and the melting point (594 K). An irreversible loss of the precession signal was found for both poly- and single crystals of Cd below 500 K, whereas for dilute Cd-In alloys the signal remains observed above 500 K and the amplitude shows a reversible reduction near the melting point. These experimental results are discussed in terms of a diffusion model as well as of a melting model.

To examine the orientation and magnitude of the electric field gradient (EFG) created by a vacancy at the111In probe site, PAC spectra were measured in a Cd single crystal as a function of the crystal orientation after a quenching and aging treatment at 120 K. The results are compared with calculated spectra based on a point charge model of the EFG created by a vacancy at the site of the probe atom.

NMR-ON measurements have been performed for60CoFe−Si. The samples of Fe−Si (6 at. %) single crystal dish with the (110) surface were used. Rosonance measurements have been carried out with the magnetization direction along the and the axes. Five prominent resonances were found at 166, 162, 151, 147 and 135 Mllz. The resonance at 166 Mllz has been known to be due to60Co without neighbourt Si atoms. The resonances at 151 and 135 Mllz are due to60Co nuclei with one and two Si atom(s) in the 1st neighbour site, respectively. The resonances at 162 and 147 Mllz would be due to the contribution of the 3rd and the magnetization axes, the differences of the resonance width have been qualitatively explained using the dipolar field.

Myelodysplastic syndrome (MDS) with unbalanced t(1;7) after severe anemia (SAA) in childhood as a variant form of monosomy 7

Novel missense mutations in the tyrosine kinase domain of the platelet-derived growth factor receptor α ( PDGFRA ) gene in childhood acute myeloid leukemia with t(8;21)(q22;q22) or inv(16)(p13q22)

Forty-four infants with acute lymphoblastic leukemia (ALL) characterized by MLL gene rearrangements were treated on a protocol of intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT) between November 1998 and June 2002. The remission induction rate was 91.0%, and the 3-year overall survival and event-free survival (EFS) rates, with 95% confidence intervals, were 58.2% (43.5%-72.9%) and 43.6% (28.5%-58.7%), respectively. Univariate analysis of EFS by presenting features indicated a poorer outcome in patients younger than 6 months of age with high white blood cell counts (/= 100 x 10(9)/L; EFS rate, 9.4% versus 55.1% for all others, P = .0036) and in those with central nervous system invasion (EFS rate, 10.0% versus 56.9% for all others, P = .0073). The 3-year posttransplantation EFS rate for the 29 patients who underwent HSCT in first remission was 64.4% (46.4%-82.4%). In this subgroup, only the timing of HSCT (first remission versus others) was a significant risk factor by multivariate analysis (P.0001). These results suggest that early introduction of HSCT, possibly with a less toxic conditioning regimen, may improve the prognosis for infants with MLL(+) ALL. Identification of subgroups or patients who respond well to intensified chemotherapy alone should have a high priority in future investigations.

Mutation of the AML1/RUNX1 gene in a transient myeloproliferative disorder patient with Down syndrome

Epstein-Barr virus (EBV) infection has been associated with infectious mononucleosis, EBV-associated hemophagocytic syndrome (EBV-AHS), chronic active EBV infection (CAEBV), lymphomas, inflammatory pseudotumor, lymphomatoid granulomatosis, and nasopharyngeal carcinoma. EBV-AHS and CAEBV are more lethal than infectious mononucleosis with imaging findings of gallbladder wall thickening, pleural effusion, cardiomegaly, and hepatomegaly. EBV infection is also associated with benign and malignant tumors.

The objectives were as follows: Firstly, to estimate the overall probability of event-free survival (EFS) and isolated CNS relapse in the studies for children with acute lymphoblastic leukemia (ALL) during the 1980s and 1990s. Secondly, to report the EFS according to presenting features and lineage. Thirdly, to evaluate the treatment results re-classified by the risks of NCI criteria. Four consecutive protocol studies were performed in the Tokyo Children's Cancer Study Group: L81-10 protocol (1981-1984, 189 patients), L84-11 (1984-1989, 484 patents), L89-12 (1989-1992, 418 patients) and L92-13 (1992-1995, 347 patients). Overall EFS at 5 years in each protocol was 56.5 +/- 3.8(1 s.e.)%, 71.0 +/- 2.1%, 67.8 +/- 2.3%, and 63.4 +/- 2.7%, respectively. The cumulative isolated CNS relapse rate at 5 years was 8.1 +/- 2.1%, 3.5 +/- 0.9%, 3.6 +/- 1.0%, 1.0 +/- 0.6. The EFS in SR/HR (standard risk/high risk) according to the NCI criteria in B-precursor ALL at 5 years was 61.9 +/- 4.3%/41.4 +/- 7.4% (lineage was not confirmed.), 72.5 +/- 2.6%/63.4 +/- 5.0%, 77.4 +/- 2.7%/56.3 +/- 4.7%, and 67.8 +/- 3.4%/56.7 +/- 5.4% in each protocol. Also EFSs according to NCI SR/HR at 5 years of T-ALL in protocols L84-11, L89-12 and L92-13 were 55.6 +/- 16.6%/60.9 +/- 10.1%, 72.7 +/- 13.4%/51.6 +/- 9.1%, and 77.1 +/- 14.4%/53.6/10.1%, respectively. The truncation of maintenance therapy to 6 months resulted in a decreased EFS in L92-13, particularly due to an increase of bone marrow relapse after cessation of therapy in SR and HR. The NCI risk criteria work properly even in the patients treated by different intensities, so that it makes the comparison possible among the patients in various groups. The overall EFSs in childhood ALL improved in 1980s, but it seemed stable or decreased in 1990s. The short maintenance therapy resulted in poor outcome in SR on the L92-13 protocol. Many of these late relapsers were effectively rescued and overall survival remained at a high level. The proportion of patients who received cranial irradiation reduced without any increase of the CNS events.

Background: The role of laparoscopy in children with cancer has not been fully defined. The aims of this study were to develop an optimal surgical procedure for the treatment of abdominal neuroblastoma in the laparoscopic era and to evaluate the advantages and disadvantages of laparoscopy in the pediatric population. Methods: Since July 1997, 37 children were diagnosed with abdominal neuroblastoma at our center, and 44 surgical procedures were performed on them. Patients with advanced neuroblastoma underwent laparoscopic biopsy, open biopsy, and delayed primary or second-look excision, whereas early neuroblastoma cases had either laparoscopic or open excision. We compared the length of the operation, intraoperative blood loss, length of hospital stay, complications, and time to start postoperative feeding and chemotherapy for the laparoscopic and open surgery groups. Results: Length of stay and time to postoperative feeding and chemotherapy were significantly lower in the laparoscopic group than the open surgery group. However, there were no significant differences between the two groups in length of operation and intraoperative blood loss. Conclusion: Laparoscopic biopsy and excision of abdominal neuroblastoma are effective and efficient surgical procedures in children.

The purpose of this study was to investigate the biological characteristics and prognostic value of in vitro three-drug resistance to prednisolone, L-asparaginase, and vincristine in childhood acute lymphoblastic leukemia (ALL). We carried out in vitro tests with a 4-day culture and a methyl-thiazol-tetrazolium assay on bone marrow samples from 209 children newly diagnosed with ALL. After testing the resistance of leukemic cells to 14 drugs, we classified the patients into two groups according to their sensitivity to three drugs (prednisolone, L-asparaginase, and vincristine) used in remission induction therapy. The three-drug resistant group (RR: sensitive to no drugs or to one drug) correlated with both short-term and long-term treatment failure. Three-year event-free survival (95% confidence interval) for the sensitive group (SS: sensitive to two or three drugs) was 0.813 (0.773-0.853) and that of the RR group was 0.616 (0.569-0.669) (P = 0.0001). Univariate analysis showed that Philadelphia-chromosome (Ph1) positivity and immunophenotype of mixed lineage were also prognostic factors in the 209 patients. The prognosis of the SS/RR drug resistance profile within 14 Ph1 patients was marginally significant (P = 0.062). Multivariate Cox regression analysis showed that Ph1 was an overwhelmingly adverse factor in event-free survival, with a relative hazard of 5.37 (2.57-11.21, P0.0001), followed by RR, with a relative hazard of 2.98 (1.69-5.25, P = 0.0001). Furthermore, we clarified the characteristics of the RR group by examination of the pattern of drug resistance to other drugs in comparison with the SS group. The leukemic cells of RR patients were more resistant than those of SS patients (P0.0001) to all the drugs tested, with resistance ratios of 1.6 to 13.1 (mean 3.4). In conclusion, in vitro three-drug resistance at the initial stage is an important independent predictor of treatment failure for both induction response and long-term outcome in childhood ALL.

Three patient with hemophilia B who developed anti-factor IX antibodies were reported. All 3 had allergic and/or anaphylactoid symptoms when the antibodies were found. The antibodies were noted between 4 and 17 days after exposure to factor IX. It was suggested that the anaphylactoid symptoms were closely related to the occurrence of anti-factor IX antibodies.

Hearing impairment is not often considered as a potential complication of general anesthesia, despite several reports of post-operative sensorineural hearing loss. These disorders have occurred after otological as well as cardiobypass surgery. We experienced two patients both of whom had undergone orthopedic surgery. In both cases the patients experienced bilateral reversible hearing impairment after general anesthesia with nitrous oxide. It is likely that a change in the middle ear pressure as a result of Eustachian tube dysfunction may have caused transient conductive hearing loss added to sensorineural hearing disorder. After these cases we interviewed a series of 115 patients who had undergone general anesthesia to assess the extent of this problem. Contrary to our expectation, 7 patients complained of ear fullness or autophony after inhalation of nitrous oxide, although these symptons diminished within 24 hours. It is important to be aware of the possibility of hearing impairment when nitrous oxide is used especially if the patient has a history of a previous middle ear disease.

We examined mRNA expression and internal tandem duplication of the Fms-like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase-polymerase chain reaction (RT-PCR) and genomic PCR followed by sequencing. By RT-PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B-precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B-ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

N822 mutation of KIT gene was frequent in pediatric acute myeloid leukemia patients with t(8;21) in Japan: a study of the Japanese childhood AML cooperative study group

Recurrent translocation t(10;11) has been reported to be associated with acute myeloid leukemia (AML). Recently, two types of chimeric transcripts, MLL-AF10 in t(10;11)(p12;q23) and CALM-AF10 in t(10;11)(p13;q14), were isolated. t(10;11) is strongly associated with complex translocations, including invins(10;11) and inv(11)t(10;11), because the direction of transcription of AF10 is telomere to centromere. We analyzed a patient of AML with t(10;11)(p11.2;q23) and identified ABI-1 on chromosome 10p11.2, a human homolog to mouse Abl-interactor 1 (Abi-1), fused with MLL. Whereas the ABI-1 gene bears no homology with the partner genes of MLL previously described, the ABI-1 protein exhibits sequence similarity to protein of homeotic genes, contains several polyproline stretches, and includes a src homology 3 (SH3) domain at the C-terminus that is required for binding to Abl proteins in mouse Abi-1 protein. Recently, e3B1, an eps8 SH3 binding protein 1, was also isolated as a human homolog to mouse Abi-1. Three types of transcripts of ABI-1 gene were expressed in normal peripheral blood. Although e3B1 was considered to be a full-length ABI-1, the MLL-ABI-1 fusion transcript in this patient was formed by an alternatively spliced ABI-1. Others have shown that mouse Abi-1 suppresses v-ABL transforming activity and that e3B1, full-length ABI-1, regulates cell growth. In-frame MLL-ABI-1 fusion transcripts combine the MLL AT-hook motifs and DNA methyltransferase homology region with the homeodomain homologous region, polyproline stretches, and SH3 domain of alternatively spliced transcript of ABI-1. Our results suggest that the ABI-1 gene plays a role in leukemogenesis by translocating to MLL.

Site-specific recombination of the TAL1 gene was analyzed by Southern blotting and polymerase chain reaction (PCR) in 44 cases of childhood T-cell acute lymphoblastic leukemia (T-ALL), 20 cases of childhood T-cell non-Hodgkin's lymphoma (T-NHL) and 35 cases of adult T-cell malignancies. This recombination was found in 10 (22.7%) of 44 childhood T-ALL patients, but in none of the T-NHL or adult T-cell malignancies. Recombination of the TAL1 gene was therefore suggested to be specific for childhood T-ALL. The immunophenotypic features of the 10 T-ALL patients with this recombination were CD1-, CD2+, CD4-, CD7+, CD10-, and they had a significantly better outcome than other T-ALL cases without the recombination. The PCR technique revealed minimal residual disease (MRD) in 2 patients. One showed persistent MRD, while in the other MRD was recognized only at initial diagnosis. Further investigation is needed whether T-ALL with this recombination constitutes a distinct clinical subgroup among childhood T-ALL patients.

Correlations between alterations of the p53 gene and clinical features were examined in childhood acute lymphoblastic leukemia (ALL). We analyzed 147 patients and 38 cell lines for p53 mutations within exons 5 to 9 (2 to 11 in some of them) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. p53 gene mutations were found in 3 of 62 (5%) patients at diagnosis, 1 of 14 (7%) patients at relapse, and 13 of 20 (65%) cell lines in T-ALL, 2 of 20 (10%) patients at diagnosis, 4 of 4 (100%) patients at relapse, and 4 of 5 (80%) cell lines in t(1;19)-ALL, 1 of 23 (4%) patients at diagnosis, 2 of 22 (9%) patients at relapse, and 5 of 12 (42%) cell lines in common ALL other than t(1;19) or t(9;22)-ALL and 3 of 3 (100%) patients at diagnosis in B-ALL. In t(1;19)-ALL, p53 gene alterations were associated with a poor prognosis. The patients with p53 mutations had a trend towards poor prognosis in childhood ALL without B-ALL. p53 gene mutation is not always associated with the current prognostic factors. This alteration may become one of the important prognostic factors, if the detection of a small number of the leukemic cells with the p53 gene mutation would be possible.

Loss of heterozygosity (LOH) on chromosome 18q21 is found frequently in various human cancers. Three candidate tumor suppressor genes, DCC (deleted in colorectal carcinomas), DPC4 (deleted in pancreatic carcinomas, locus 4), and MADR2/JV18-1 (MAD-related gene 2), have been cloned and identified from this chromosome region. We have reported recently that LOH on chromosome 18q is observed frequently in neuroblastoma. Alterations of DCC are involved in many human tumors. DPC4 and MADR2/JV18-1 are recently demonstrated to be altered in pancreatic and colorectal cancers, respectively. To confirm if inactivation of the DCC, DPC4, and MADR2/JV18-1 genes is involved in the pathogenesis of neuroblastoma and to clarify the mechanism of inactivation, we analyzed the expression of DCC, DPC4, and MADR2/JV18-1 in neuroblastoma cell lines and primary tumors by reverse transcription-PCR and investigated the mutations in the coding regions of these genes by PCR/reverse transcription-PCR single-strand conformation polymorphism. We found that 12 of 25 (48%) cell lines and 14 of 32 (44%) primary tumors, including 3 with 18q LOH, had absent or reduced expression of DCC mRNA. Expression was more likely to be reduced in advanced (67%) than in early stage neuroblastomas (24%) (P = 0.036), suggesting that inactivation of the DCC gene plays an important role in the progression of neuroblastoma. Altered expression of DPC4 was found in six (24%) cell lines and six (19%) tumors. MADR2/JV18-1 expression was reduced or absent only in four (16%) cell lines and three (9%) tumors. Mutations of the DCC genes were examined in 25 of 29 exons in neuroblastoma cell lines, and those exons in which mutations were found were further examined in primary tumors. We found missense mutations of AAC (Asn) to AGC (Ser) at DCC codon 176 in one cell line and ACC (Thr) to ATC (Ile) at codon 1105 in one cell line and tumor, respectively; polymorphisms of CGA (Arg) to GGA (Gly) at codon 201 and TTT (Phe) to TTG (Leu) at codon 951 in most of the cell lines and tumors; and a silent mutation of GAG (Glu) to GAA (Glu) at codon 118 in four cell lines and five primary tumors. We did not identify any mutations in the DPC4 and MADR2/JV18-1 genes in neuroblastoma. Our results suggested that mutations of the DCC gene may be involved in the pathogenesis of neuroblastomas but failed to account for the relatively high frequency of the altered expression, implying that other mechanisms are responsible for the inactivation of the DCC gene in neuroblastoma. Low frequency of reduced or absent mRNA expression and lack of mutations in DPC4 and MADR2/JV18-1 genes suggested a limited role for these two genes in neuroblastoma.

We reported previously that loss of heterozygosity (LOH) on chromosomes 2q, 9p and 18q frequently occurs in neuroblastoma and that patients with 9p LOH in the tumors showed statistically significant association with an advanced stage of the disease and poor prognosis. To determine the role of chromosome 9 loss in neuroblastoma, we performed deletion mapping of chromosome 9 in 80 cases of neuroblastoma using 11 polymorphic microsatellite markers and a restriction fragment length porymorphism marker. LOH at one or more loci on chromosome 9 was detected in 33 of 80 cases (41%). Chromosome 9p was lost in 24 of 80 cases (32%), whereas chromosome 9q was lost in 18 of 80 cases (23%). There were two commonly deleted regions mapped to 9p21 between the D9S171 marker and the IFNB1 marker and 9q34-qter distal to the D9S176 marker. In addition, patients with LOH at 9p21 but not at 9q34-qter in the tumors showed statistically significant association with poor prognosis (P = 0.023). Because the commonly deleted regions at 9p21 includes the p16 (CDKN2A) gene, the status of the p16 gene was further examined in 80 fresh tumors and 19 cell lines of neuroblastoma. A missense mutation was detected at codon 52 in a fresh tumor. The p16 gene was not expressed in 13 of 19 cell lines (72%), and 5 of the 13 cell lines displayed methylation of the CpG island surrounding the first exon of the p16 gene. These results suggest that the p16 gene is a candidate tumor suppressor gene for neuroblastoma, and its inactivation may contribute to the progression of neuroblastoma.

A 9-year-old with chronic myelogenous leukemia (CML) who received bone marrow transplantation from an unrelated donor (UBMT), is reported. He also suffered from congenital heart disease (CHD) consisting of corrected transposition of the great arteries and dextrocardia. The cardiac output was within normal limits. The conditioning regimen included busulfan, melphalan, ALG and TLI. Cyclophosphamide was not used because of it cardiotoxicity. The HLA-phenotype of the donor was identical with that of the patient. DNA typing showed was haplodentical DRB1. The patient is alive 18 months after the UBMT. This case showed that UBMT was possible in a CML patient with CHD, without congestive heart failure.

We analyzed 60 B precursor acute lymphoblastic leukemia (ALL) primary samples and 15 cell lines for homozygous deletions of p16 and p15 genes and mutations of p16 gene. These included five cell lines and 13 primary samples with the t(1;19)(q23;pl3), and eight primary samples with the t(9;22)(q34;qll). Of 10 cell lines without t(1;19), homozygous deletion of both p16 and p15 genes was found in eight cell lines (80%), and a rearrangement of p16 in one cell line (10%). In contrast, only one (20%) of the five cell lines with t(1;19) showed homozygous deletion or rearrangement of p16/p15 gene. Thirteen of 60 (22%) primary samples demonstrated p16 gene homozygous deletion. No case with t(1;19) showed homozygous deletion of p16 gene (0/13, 0%), while cases without t(1;19) showed considerable incidence of p16 gene homozygous deletion (13/47, 28%). These results suggest that the incidence of deletions of p16 gene differs according to the subtypes of B precursor ALL. We also compared the frequency of p16 gene homozygous deletion between the patients at diagnosis and at relapse. Nine of 45 (20%) samples at diagnosis and four of 22 (18%) samples at relapse showed p16 homozygous deletions. The similarity of the rate in these two groups raises the question of the role of p16 gene in progression of B precursor ALL. Mutations were found in three of the primary cases (5%); the mutations included two nonsense mutations at codon 72 and one missense mutation at codon 98. All the mutations found in this study were heterozygous, and the clinical relevance of p16 gene mutation is yet to be determined in these case

We have investigated the mutation of the TP53 gene in hepatoblastomas (HBLs) by using polymerase chain reaction-single strand conformation polymorphism and direct sequencing in 38 HBL tumor samples and in two HBL cell lines. We detected the TP53 gene mutation in an anaplastic hepatoblastoma cell line, but no aberration of the TP53 gene (exons 5-9) was found in tumor samples and in the other HBL cell line. The mutation of the cell line was a missense mutation from GAC (asparagine) to CAC (histidine) at codon 281, which was different from the G-to-T transversion of codon 249 that is frequently found in adult hepatocellular carcinomas (HCCs). In addition, we performed Southern blot analysis of the MDM2 gene, but we did not find MDM2 gene amplification in 19 cases tested. Our results suggest that, in contrast to the findings in HCCs in adults, TP53 gene aberrations are not involved in the development or progression of HBLs in children.

Although relatively high incidence of loss of heterozygosity (LOH) on chromosomes 1p, 11q, and 14q have been reported in neuroblastoma, it is still unclear whether or not LOH occurs specifically on these chromosome arms in neuroblastoma since only a few chromosomal arms have been examined for LOH in previous studies. Therefore, we screened 81 cases of tumors for LOH on all 22 autosomes and chromosome X using 35 restriction fragment length polymorphism markers and eight microsatellite markers. High incidence of LOH (20%) was observed on six chromosome arms; 1p (26%), 2q (30%), 9p (36%), 11q (24%), 14q (22%), and 18q (31%). Frequencies of LOH on other chromosome arms were less than 13%. Patients with 9p LOH in the tumors showed statistically significant association with advanced stage of the disease and poor prognosis (P = 0.037 and P = 0.003, respectively) independently from N-myc amplification, while LOH on other chromosomes did not show association with stage, prognosis, and N-myc amplification. Thus, besides LOH on chromosomes 1p, 11q, and 14q, LOH on chromosomes 2q, 9p, and 18q also occurs relatively frequently in neuroblastoma, indicating the involvement of multiple tumor suppressor genes in the development of neuroblastoma. It is possible that there is a novel tumor suppressor gene on chromosome 9p which is involved in the progression of neuroblastoma.