CCL22 mutations in large granular lymphocytic leukemia.

Not available.

Immature Platelet Fraction and Its Kinetics in Neonates.

Thrombocytopenia is a common abnormality encountered in the neonatal period, and immature platelet fraction (IPF) may be an informative indicator of thrombopoiesis; however, data on IPF in neonates are scarce. To define reference intervals (RIs) and factors affecting IPF in neonates, we measured the IPF of 533 consecutive neonates. With a multiple regression analysis of 330 newborns with normal platelet counts at birth, premature delivery, neonatal asphyxia, intrauterine infection, chromosomal abnormalities, and respiratory disorders were identified as independent factors for IPF%. The RIs of IPF% and absolute IPF value in neonates were determined to be 1.3% to 5.7% and 3.2 to 14.5×10 9 /L, respectively. On day 14 after birth, IPF% increased to twice the value at birth and thereafter returned to the previous value on day 28. Reticulocyte counts, in contrast, were the lowest at day 14. IPF% was increased in 16 thrombocytopenic patients with various clinical conditions, especially those with immune-mediated thrombocytopenia. IPF in neonates may be evaluated essentially based on the same RIs as in adults, although some precautions must be taken when evaluating IPF in neonates in the first 2 weeks of life. IPF may be useful for evaluating thrombopoiesis and thrombocytopenia in neonates.

A Novel Amino Acid Substitution, Fibrinogen Bßp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion.

We identified a novel heterozygous variant, Bßp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bßp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bßp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bßγ complex in Bßp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bßp.P234 residue is located in the contact region between the Bß and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bßp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bß and γ chains impact the assembly and secretion of fibrinogen.

Safety and tolerability of allogeneic dendritic cell vaccination with induction of Wilms tumor 1-specific T cells in a pediatric donor and pediatric patient with relapsed leukemia: a case report and review of the literature.

A 15-year-old girl with acute lymphoblastic leukemia received allogeneic dendritic cell vaccination, pulsed with Wilms tumor 1 (WT1) peptide, after her third hematopoietic stem cell transplantation (HSCT). The vaccines were generated from the third HSCT donor, who was her younger sister, age 12 years. The patient received 14 vaccines and had no graft-versus-host disease or systemic adverse effect, aside from grade 2 skin reaction at the injection site. WT1-specific immune responses were detected after vaccination by both WT1-tetramer analysis and enzyme-linked immunosorbent spot assay. This strategy may be safe, tolerable and even feasible for patients with a relapse after HSCT.

Efficacy of Touch Imprint Cytology in Intraoperative Diagnosis of Invasive Mucinous Adenocarcinoma of the Lung: A Case Report and Literature Review.

A preoperative diagnosis of the peripheral small lung nodule is often difficult, and an intraoperative frozen section diagnosis (FSD) is performed to guide treatment strategy. However, invasive mucinous adenocarcinoma (IMA) is prone to be overlooked because of the low sample quality and weak atypia. We herein report a case of IMA, in which touch imprint cytology (TIC) revealed diagnostic efficacy. A 74-year-old male with a small, subsolid nodule in the right upper lobe underwent a thoracoscopic wedge resection. A grayish brown, 10 × 7 mm-sized nodule was observed on the cut surface. Intraoperative FSD revealed lung tissue with mild alveolar septal thickening and stromal fibrosis but without overt atypia. Meanwhile, TIC revealed mucus and a few epithelial cells with intranuclear inclusions, which pathologists evaluated as reactive. Finally, focal organizing pneumonia was tentatively diagnosed, and surgery was finished without any additional resection. However, permanent section diagnosis revealed a microinvasive mucinous adenocarcinoma. Nuclear inclusions were confirmed in tumor cells. In the intraoperative setting, TIC may be more advantageous than FSD in observing nuclear inclusions and mucus. Mucinous background and nuclear inclusion on TIC may suggest IMA even if FSD does not suggest malignancy in an intraoperative diagnosis of the peripheral small lung nodule.

Quantitative evaluation of citrullinated fibrinogen for detection of neutrophil extracellular traps.

Activated neutrophils release neutrophil extracellular traps (NETs) composed of chromatin filaments containing bactericidal proteins and enzymes. This process, known as NETosis, is an innate host defense mechanism. However, NET accumulation can lead to uncontrolled inflammation and organ damage. Therefore, NET detection provides clinically important information for the assessment of inflammatory conditions. We investigated whether quantification of citrullinated fibrinogen (C-Fbg), which is catalyzed by peptidylarginine deiminase (PAD) released during NETosis, can be used to detect NETs. Human neutrophils were stimulated with fibrinogen using phorbol 12-myristate 13-acetate (PMA). The myeloperoxidase (MPO)-DNA complex and C-Fbg concentrations in the culture supernatants were quantified using an enzyme-linked immunosorbent assay. The protein levels of peptidylarginine deiminase 2 and 4 in culture supernatants and mRNA levels in PMA-stimulated neutrophils were also assessed. The levels of the MPO-DNA complex in the supernatants of PMA-stimulated neutrophils increased, indicating NETosis. C-Fbg level also increased, which was suppressed by both NETosis and PAD inhibitors. PAD2 was detected in the culture supernatant; however, PAD4, but not PAD2, mRNA levels increased in PMA-stimulated neutrophils. This study quantitatively demonstrates that fibrinogen is citrullinated by PAD derived from PMA-stimulated neutrophils upon NETosis. Although further studies are needed for clinical application, quantification of C-Fbg in blood may help detect the presence of NETs.

An unusual cytological presentation of solid pseudopapillary neoplasm of the pancreas mimicking adenoid cystic carcinoma: a case report and literature review.

Endoscopic ultrasound-guided fine-needle aspiration has become the common procedure for the diagnosis of pancreatic mass, and cytological examination is usually the first approach. Solid pseudopapillary neoplasm (SPN) cytologically represents papillary structures of branching capillaries surrounded by discohesive neoplastic cells. However, it may present various degrees of tissue degeneration, causing diagnostic challenges. Here, we report a 21-year-old female who had a 2-cm-sized mass in the pancreas head. Cytological examination revealed clumps of small round/oval cells that represented microcystic configurations with mucus, mimicking adenoid cystic carcinoma or mucinous adenocarcinoma. Cercariform cells, nuclear grooves/folding, and cytoplasmic vacuoles were not observed. Histopathological examination revealed confluent small glandular structures containing acidic mucus. The tumor cells were positively stained for ß-catenin, CD10, and CD56, and negative for chromogranin A and E-cadherin, suggesting SPN, micropseudocystic variant. This variant has been scarcely described, but we should recognize it for accurate cytological triage of pancreatic tumors.

Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis.

Primary tumor cells metastasize to a distant preferred organ. However, the most decisive host factors that determine the precise locations of metastases in cancer patients remain unknown. We have demonstrated that post-translational citrullination of fibrinogen creates a metastatic niche in the vulnerable spots. Pulmonary endothelial cells mediate the citrullination of fibrinogen, changing its conformation, surface charge, and binding properties with serum amyloid A proteins (SAAs), to make it a host tissue-derived metastatic pathogen. The human-specific SAAs-citrullinated fibrinogen (CitFbg) complex recruits cancer cells to form a protein-metastatic cell aggregation in humanized SAA cluster mice. Furthermore, a CitFbg peptide works as a competitive inhibitor to block the homing of metastatic cells into the SAAs-CitFbg sites. The potential metastatic sites in the lungs of patients are clearly visualized by our specific antibody for CitFbg. Thus, CitFbg deposition displays metastatic risks for cancer patients, and the citrullinated peptide is a new type of metastasis inhibitor.

T cell clonal expansion and STAT3 mutations: a characteristic feature of acquired chronic T cell-mediated pure red cell aplasia.

Acquired chronic pure red cell aplasia (PRCA) develops idiopathically or in association with other medical conditions, including T cell large granular lymphocytic leukemia (T-LGLL) and thymoma. T cell dysregulation is considered a cardinal pathogenesis of PRCA, but genetic-phenotypic associations in T cell abnormalities are largely unclear. We evaluated an extended cohort of 90 patients with acquired PRCA, including 26 with idiopathic, 36 with T-LGLL-associated and 15 with thymoma-associated PRCA, for their T cell immuno-phenotypes, clonalities and STAT3 mutations. TCR repertoire skewing of CD8+ T cells was detected in 37.5% of idiopathic, 66.7% of T-LGLL-associated and 25% of thymoma-associated PRCA patients, and restriction to Vß1 was most prominent (41%). Clonalities of TCRß or γ chain and STAT3 mutational status were statistically associated (P = 0.0398), and they were detected in all three subtypes. The overall response rate to cyclosporin A was 73.9%, without significant difference by subtypes nor STAT3 mutational status. The T cell dysregulations, such as TCR repertoire skewing with predominant Vß1 usage, clonality and STAT3 mutations, were frequently found across the subtypes, and the close associations between them suggest that these T cell derangements reflect a common pathophysiological mechanism among these PRCA subtypes.

Novel variant fibrinogen γp.C352R produced hypodysfibrinogenemia leading to a bleeding episode and failure of infertility treatment.

INTRODUCTION: We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder. MATERIALS AND METHODS: Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting. RESULTS: Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells. CONCLUSION: We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.

Changes in serum citrullinated fibrinogen concentration associated with the phase of bacteremia patients.

BACKGROUND: Citrullinated fibrinogen (C-Fbg) has been detected in rheumatoid arthritis; however, few studies have reported the role of C-Fbg in other inflammatory diseases. This study aimed to clarify the changes in serum C-Fbg associated with the bacteremia phase. METHODS: We measured serum C-Fbg concentration in bacteremia patients. C-Fbg levels at each phase of bacteremia, classified by white blood cell (WBC) count and neutrophil left shift change, were compared with those of healthy control (HC). The correlation between C-Fbg concentration and certain inflammatory markers, or citrullinated histone H3 concentration was assessed. Multiple linear regression (MLR) analysis was used to examine the association of log C-Fbg with certain inflammatory markers. RESULT: Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p < 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p < 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p < 0.01). CONCLUSION: Serum C-Fbg levels increased in bacteremia patients, and this was consistent with an influx of neutrophils into the blood stream in accordance with the bacteremia phase.

Congenital fibrinogen disorder with a compound heterozygote possessing two novel FGB mutations, one qualitative and the other quantitative.

INTRODUCTION: Congenital fibrinogen disorders result from genetic mutations in FGA, FGB, or FGG resulting in quantitative fibrinogen deficiencies (afibrinogenemia or hypofibrinogenemia) or qualitative fibrinogen deficiencies (dysfibrinogenemia). Hypodysfibrinogenemia sharing features with hypo- and dysfibrinogenemia is rare. We performed genetic and functional analyses of a 31-year-old woman with suspected hypodysfibrinogenemia. MATERIALS AND METHODS: Functional and antigenic fibrinogen values of patient were 1.05 and 1.24 g/L, respectively. DNA sequence and western blotting analyses for plasma fibrinogen were performed. A minigene incorporating the mutational region was transfected into a Chinese hamster ovary cell line (CHO), and reverse transcription products were analyzed. Assembly and secretion were examined using the recombinant variant fibrinogen. We purified the patient's plasma fibrinogen and analyzed thrombin-catalyzed fibrin polymerization (TCFP). RESULTS AND CONCLUSIONS: DNA sequencing revealed compound heterozygous nucleotide mutations with FGB 35 bp c.1245-17_1262 or -16_1263 del and FGB c.510T>A (resulting in Bßp.N170K substitution) on different alleles. We did not detect shortened Bß-chain peptides in the plasma using western blotting analysis. A minigene incorporating the deletion DNA showed two aberrant mRNA products. The secretion of Bßp.N170K-fibrinogen-CHO was almost same as normal Bß-fibrinogen-CHO. TCFP of plasma Bßp.N170K fibrinogen was slightly lower than that of normal plasma fibrinogen. Aberrant splicing products derived from the 35 bp deletion caused hypofibrinogenemia due to nonsense-mediated mRNA decay and suggested the presence of only Bßp.N170K fibrinogen in patient's plasma. Bßp.N170K caused dysfibrinogenemia due to a delay in lateral aggregation. These findings demonstrated that these mutations respectively affected the fibrinogen quality and quantity, resulting in hypodysfibrinogenemia.

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens.

INTRODUCTION: Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. METHODS: Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. RESULTS: The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. CONCLUSION: The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and "D:D" interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia.

Comparison of molecular structure and fibrin polymerization between two Bß-chain N-terminal region fibrinogen variants, Bßp.G45C and Bßp.R74C.

We identified two heterozygous dysfibrinogenemias, Bßp.Gly45Cys (Kyoto VII; K-VII) and Bßp.Arg74Cys (Iida II; I-II). The impairment of polymerization of Bßp.G45C has been well analyzed; however, that of Bßp.R74C has not. Thus, we compared fibrin polymerization between these variants. To determine the structural and functional characterization of purified fibrinogens, we performed immunoblotting analysis, kinetic analyses of fibrinopeptide A and B release, and thrombin- or batroxobin-catalyzed fibrin or fibrin monomer polymerization. Immunoblotting analysis showed that both variant fibrinogens had variant fibrinogen-albumin complexes and variant fibrinogen multimers, and the amounts of fibrinogen-albumin complexes with fibrinogen K-VII was more than with fibrinogen I-II. Moreover, fibrinopeptide B release from fibrinogen K-VII was about 50% of the control, whereas the others were normal. The maximum slopes of polymerization for variant fibrinogens were reduced, but fibrinogen K-VII was reduced more than fibrinogen I-II. The present study demonstrated that both Bßp.G45C and Bßp.R74C variants showed the presence of variant fibrinogen-albumin complexes and variant fibrinogen multimers, and polymerization of Bßp.G45C was impaired more than Bßp.R74C. Our study and several previous reports concerning the clinical phenotype of both variants suggested the risks of bleeding for patients with Bßp.G45C and thrombosis for patients with Bßp.R74C.

Leaf shape deters plant processing by an herbivorous weevil.

The shapes of plant leaves are remarkably diverse, but their ecological functions are largely unknown. Reports on the effects of leaf shape on biotic interactions such as herbivory are especially scarce, partly because herbivorous insects rarely rely on leaf shape for host selection. Here, we show that leaf shape acts as a physical deterrent against a leaf-processing herbivore. Plants in the genus Isodon (Lamiaceae) host a specialized leaf-rolling weevil (Apoderus praecellens) whose ovipositing females process an entire leaf into a leaf roll to serve as larval food and shelter. Among the species of Isodon, I. umbrosus var. hakusanensis is exceptional in that it has deeply lobed leaves. Because leaf processing follows a consistent sequence of complex behaviours, the unusual shape of I. umbrosus leaves may disrupt this process. Under both natural and laboratory conditions, female weevils preferred I. trichocarpus, a close relative with non-lobed leaves, over I. umbrosus. Nutritional properties of the leaves do not explain this preference because weevil larvae developed equally well on both hosts. Modifying the non-lobed I. trichocarpus leaves to mimic the shape of I. umbrosus leaves also discouraged leaf processing. Leaf processing often terminated because weevils failed to complete the inspection routine on I. umbrosus leaves. Leaf shape may be an important but overlooked factor that affects the interactions between plants and leaf-processing herbivores.

γD318Y fibrinogen shows no fibrin polymerization due to defective "A-a" and "B-b" interactions, whereas that of γK321E fibrinogen is nearly normal.

BACKGROUND: The fibrinogen γ-module has several functional sites and plays a role in dysfibrinogenemia, which is characterized by impaired fibrin polymerization. Variants, including γD318Y and γΔN319D320, have been reported at the high affinity Ca2+-binding site, and analyses using recombinant fibrinogen revealed the importance of this site for fibrinogen functions and secretion. We examined the polymerization abilities of the recombinant fibrinogen variants, γD318Y and γK321E. MATERIALS AND METHODS: γD318Y and γK321E were produced using CHO cells and fibrinogen functions were examined using thrombin- or batroxobin-catalyzed polymerization, gel chromatography, protection against plasmin degradation, and factor XIIIa cross-linking. RESULTS: γD318Y did not show any polymerization by thrombin or batroxobin, similar to γΔN319D320, whereas γK321E had slightly impaired polymerization. The functions of Ca2+ binding, hole 'a', and the "D-D" interaction were markedly reduced in γD318Y, and gel chromatography suggested altered protofibril formation. In silico analyses revealed that structural changes in the γ-module of these variants were inconsistent with polymerization results. The degree of structural changes in γD318Y was moderate relative to those in γD318A and γD320A, which had markedly impaired polymerization, and γK321E, which showed slightly impaired polymerization. CONCLUSION: Our results suggest that no polymerization of γD318Y or γΔN319D320 was due to the loss of both "A-a" and "B-b" interactions. Previous studies demonstrated that "B-b" interaction alone causes polymerization of neighboring γD318A and γD320A fibrinogen, which is subsequently decreased. Marked changes in the tertiary structure of the γD318Y γ-module influenced the location and/or orientation of the adjacent ß-module, which led to impaired "B-b" interactions.

In Vivo Administration of Recombinant Human Granulocyte Colony-Stimulating Factor Increases the Immune Effectiveness of Dendritic Cell-Based Cancer Vaccination.

Significant recent advances in cancer immunotherapeutics include the vaccination of cancer patients with tumor antigen-associated peptide-pulsed dendritic cells (DCs). DC vaccines with homogeneous, mature, and functional activities are required to achieve effective acquired immunity; however, the yield of autologous monocyte-derived DCs varies in each patient. Priming with a low dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 16-18 h prior to apheresis resulted in 50% more harvested monocytes, with a significant increase in the ratio of CD11c+CD80+ DCs/apheresed monocytes. The detection of antigen-specific cytotoxic T lymphocytes after Wilms' tumor 1-pulsed DC vaccination was higher in patients treated with rhG-CSF than those who were not, based on immune monitoring using tetramer analysis. Our study is the first to report that DC vaccines for cancer immunotherapy primed with low-dose rhG-CSF are expected to achieve higher acquired immunogenicity.

[Additional chromosomal changes in impaired chimeric analysis of a patient with relapsed leukemia after bone marrow transplantation].

Chimerism analysis by polymerase chain reaction amplification of short tandem repeats (PCR-STR) has become a routine diagnostic procedure for evaluating grafts and assessing the likeliness of original disease recurrence after allogeneic stem cell transplantation. Following a sex-mismatched hematopoietic stem cell transplantation (HSCT), we monitored the clinical course of a 61-year old male AML M6 patient with trisomy 8 using PCR-STR with a TH01 locus on 11p15 and fluorescence in situ hybridization (FISH) analysis specific for alpha satellite DNA on chromosome 8. Ten months after HSCT, FISH analysis showed 24.8% recipient cells, but PCR-STR demonstrated 100% donor type chimerism. Further XY FISH analysis of May-Grünwald-Giemsa-stained bone marrow samples clearly demonstrated relapse of the original disease and G-banding analysis of bone marrow samples at relapse showed that an additional chromosomal abnormality, del(11) (p10), had deleted the PCR-STR detection site in all recipient type cells. As such, clinicians should consider the possibility that unexpected karyotype changes may invalidate PCR-STR analysis findings, especially when conflicting results appear among chimerism analyses.

Feasibility and Immune Response of WT1 Peptide Vaccination in Combination with OK-432 for Paediatric Solid Tumors.

BACKGROUND/AIM: Wilms' tumor 1 (WT1) peptide-based vaccination has been reported for its potential usefulness in targeting several cancers. The adjuvant drug OK-432 is known to have potent immunomodulation and therapeutic properties when applied in cancer treatment and may, thus, be important to trigger the appropriate immunological response in paediatric patients with a solid tumor that are vaccinated with a WT1 peptide. PATIENTS AND METHODS: Paediatric patients with a solid tumor were vaccinated with a WT1 peptide and OK-432 once every 2 weeks, for a total of seven times. RESULTS: Of the 24 patients, 18 completed the scheduled vaccinations. Sixteen patients had local skin symptoms and/or fever. In 1 patient, anaphylactic symptoms emerged at the time of the final injection, but these quickly subsided after the treatment. WT1-specific immunological responses were observed in 4 patients (22.2%). WT1 and HLA class I expression were confirmed in 100% and 85% of primary tumors, respectively. CONCLUSION: WT1 peptide vaccine therapy combined with OK-432 appears to be relatively safe for children. However further studies in a larger number of patients are necessary to confirm its safety and efficacy.