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Objective: To analyse the relationship between the preoperative systemic immune-inflammation index (SII) and the relapse-free survival (RFS) of paediatric patients with Wilms' tumour (WT) after radical surgery, and to establish and validate a prognostic survival model., Study Design: Observational study. Place and Duration of the Study: Department of Oncologic Surgery, Anhui Children's Hospital of Fudan University, Hefei, China, from January 2013 to August 2023., Methodology: A retrospective analysis was conducted on 79 WT patients treated with radical resection, with their preoperative SII values computed. The best cut-off for SII was determined through the ROC curve, categorising patients into high and low SII groups. The Kaplan-Meier method and Cox-regression were used for survival analysis. A survival prognostic model was constructed and its predictive capability gauged (AUC of the ROC)., Results: The study included 79 WT patients with a median RFS of 65 months and an average of 75.5 ± 3.4 months. The optimal cut-off value for SII was 534.95. The low SII group had a higher RFS (Log-rank: χ2 = 9.380, p = 0.002). Preoperative SII (HR = 3.277, 95% CI: 1.167 - 9.200, p = 0.024), clinical staging (HR = 8.408, 95% CI: 2.604 - 27.147, p <0.001), and tissue differentiation (HR = 2.237, 95% CI: 1.043 - 5.828, p = 0.039) were independent risk factors for RFS. The model's diagnostic performance was 0.749 (95% CI: 0.636 - 0.861). Internal validation showed an AUC of 0.723 (95% CI: 0.608 - 0.838)., Conclusion: Lower preoperative SII suggests a more favourable prognosis. The SII-based nomogram efficiently forecasts post-radical surgery prognosis for WT., Key Words: Wilms' Tumour, Systemic immune-inflammation index, Relapse-free survival, Nomogram.

Background/aim: Wilms' tumors are pediatric renal tumors that generally have a good prognosis and outcomes. Viral illnesses have been linked to development of neoplasms and should be considered as a factor that could modulate overall survival., Materials and Methods: We considered recently developed adaptive immune receptor, genomics and bioinformatics approaches to assess the potential impact of cytomegalovirus (CMV) infections in Wilms' tumor., Results: T-cell receptor (TCR) complementarity determining region-3 (CDR3) amino acid sequences from Wilms' tumor specimens represented by the Therapeutically Applicable Research to Generate Effective Treatments dataset were compared with known anti-CMV TCR CDR3s, indicating that cases representing the anti-CMV TCR CDR3s had worse outcomes. Then, a chemical complementarity scoring approach for the Wilms' tumor, TCR CDR3s and a series of CMV antigens further indicated that cases representing a higher chemical complementarity to the CMV antigens had worse outcomes., Conclusion: Overall, we present a potentially novel method to assess CMV infections and identify patients who could benefit from therapies that address such infections., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Wilms' tumor (WT) is the most common renal tumor in childhood. Pyroptosis, a type of inflammation-characterized and immune-related programmed cell death, has been extensively studied in multiple tumors. In the current study, we aim to construct a pyroptosis-related gene signature for predicting the prognosis of Wilms' tumor., Methods: We acquired RNA-seq data from TARGET kidney tumor projects for constructing a gene signature, and snRNA-seq data from GEO database for validating signature-constructing genes. Pyroptosis-related genes (PRGs) were collected from three online databases. We constructed the gene signature by Lasso Cox regression and then established a nomogram. Underlying mechanisms by which gene signature is related to overall survival states of patients were explored by immune cell infiltration analysis, differential expression analysis, and functional enrichment analysis., Results: A pyroptosis-related gene signature was constructed with 14 PRGs, which has a moderate to high predicting capacity with 1-, 3-, and 5-year area under the curve (AUC) values of 0.78, 0.80, and 0.83, respectively. A prognosis-predicting nomogram was established by gender, stage, and risk score. Tumor-infiltrating immune cells were quantified by seven algorithms, and the expression of CD8( +) T cells, B cells, Th2 cells, dendritic cells, and type 2 macrophages are positively or negatively correlated with risk score. Two single nuclear RNA-seq samples of different histology were harnessed for validation. The distribution of signature genes was identified in various cell types., Conclusions: We have established a pyroptosis-related 14-gene signature in WT. Moreover, the inherent roles of immune cells (CD8( +) T cells, B cells, Th2 cells, dendritic cells, and type 2 macrophages), functions of differentially expressed genes (tissue/organ development and intercellular communication), and status of signaling pathways (proteoglycans in cancer, signaling pathways regulating pluripotent of stem cells, and Wnt signaling pathway) have been elucidated, which might be employed as therapeutic targets in the future., (© 2024. The Author(s).)

Wilms tumor (WT) is the most common malignant tumor of the urinary system in children. Though the traditional treatment of surgery plus radiotherapy and chemotherapy achieves exciting clinical efficacy, in relapsed and refractory cases, the long-term overall survival rates are poor. Besides, chemotherapy and radiation have serious long-term toxic side effects on children. Cancer immunotherapy is a new tumor therapy that works by activating the body's immune system to allow immune cells to kill tumor cells more efficiently. Currently, cancer immunotherapy has been tested in clinical trials or basic studies in WT. This article reviews the current status of clinical trials and basic research of cancer immunotherapy in WT to promote the application of cancer immunotherapy in WT patients.

Background: Therapeutic activation of tumor-infiltrating lymphocytes using monoclonal antibodies targeting PD1 or PD-L1 (immune checkpoint inhibitors-ICIs) has revolutionized treatment of specific solid tumors in adult cancer patients, and much hope has been placed on a similar effect in relapsed or refractory solid pediatric tumors. Recent clinical trials have disappointingly shown an almost nonexistent response rate, while case reports have demonstrated that some pediatric patients do achieve durable responses when treated with this type of drug., Aim: To elucidate this paradox, we mapped the landscape of expressed neoantigens as well as the levels of immune cell infiltration in the two most common extracranial solid pediatric tumors: Wilms tumor and neuroblastoma using state-of-the-art in silico analysis of a large cohort of patients with these tumors., Methods: By integration of whole exome sequencing and RNA-sequencing, we mapped the landscape of neoantigens in the TARGET cohorts for these diagnoses and correlated these findings with known genetic prognostic markers., Results: Our analysis shows that these tumors typically have much lower levels of expressed neoantigens than commonly seen in adult cancers, but we also identify subgroups with significantly higher levels of neoantigens. For neuroblastomas, the cases with higher levels of neoantigens were confined to the group without MYCN-amplification and for Wilms tumor restricted to the TP53-mutated cases. Furthermore, we demonstrate that neuroblastomas have an unexpectedly high level of CD8+ tumor-infiltrating lymphocytes, even when compared to adult tumor types where ICI is an approved treatment., Conclusion: These results could be important to consider when designing future clinical trials of ICI treatment in pediatric patients with relapsed or refractory solid tumors., (© 2021 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

Background: Renal cancer is a common malignant tumor with an increasing incidence rate., Methods: In this study, based on the gene expression profiles, we analyzed the compositions of tumor-infiltrating immune cells (TIICs) in renal cancer and paracancerous samples using CIBERSORT. The proportions of 22 TIICs subsets in 122 paired renal carcinoma and paracancerous samples, and 224 Wilms tumor (WT) samples varied between intragroup and intergroup., Results: After analyzed the difference of TIICs composition between renal cancer and paired paracancerous samples, we found that M0 macrophages and CD8 T cells were significantly elevated, while naive B cells were significantly decreased in renal cancer samples compared with paracancerous samples. Survival analysis showed that high overall TIICs proportion, the low proportion of resting mast cells and the high proportion of activated memory CD4 T cells were associated with poor prognosis of renal cancer patients. In addition, 3 clusters were identified by hierarchical clustering analysis, and they presented a distinct prognosis. Cluster 1 had superior survival outcomes, while cluster 2 had an inferior survival outcome., Conclusions: Our study indicated that overall TIICs proportion, certain TIICs subset proportion, including resting mast cells and activated memory CD4 T cells, and distinct cluster patterns were associated with the prognosis of renal cancer, which was significant for the clinical surveillance and treatment of renal cancer.

Wilms tumor (WT) is the most common renal cancer and the most prevalent abdominal cancer in children. Children with recurrent or progressive forms of WT could benefit from novel immune-targeted approaches. While the immune status of these patients, especially the immunosuppression of peripheral T cells, was rarely reported. The present study enrolled a consecutive series of 14 Chinese WT children and 14 age- and gender-matched healthy controls. We demonstrated that plasma extracellular vesicular (EV) PD-L1 levels significantly increased in WT patients than in healthy controls. EV PD-L1 significantly inhibited the activation of human CD8 + T cells by down-regulating the cell surface CD69 expression and the intracellular IFNγ and TNFα production in vitro. In peripheral CD8 + T cells of WT patients, the intracellular IFNγ and TNFα production significantly decreased than healthy controls. The level of plasma EV PD-L1 significantly correlated with the intracellular TNFα production in peripheral CD8 + T cells of WT patients. In conclusion, the significantly increased plasma EV PD-L1 in WT patients contributed to the immunosuppression of peripheral CD8 + T cells. Monitoring the level of plasma EV PD-L1 will be helpful for the selection of immune-targeted therapies for WT patients.

The proliferation and activation of CD4+ T helper 1 (Th1) cells and CD8+ cytotoxic T lymphocytes (CTLs) that produce interferon‑γ (IFN‑γ) is an essential action of effective cancer vaccines. Recently, a novel Wilms' tumor 1 (WT1) helper peptide (WT1 HP34‑51; amino acid sequence, WAPVLDFAPPGASAYGSL) applicable for various human leukocyte antigen (HLA) subtypes (HLA‑DR, HLA‑DP and HLA‑DQ) was reported to increase peptide immunogenicity; however, the function of WT1 HP34‑51 remains unclear. In the present study, mature dendritic cells (mDCs) pulsed with WT1 HP34‑51 (mDC/WT1 HP34‑51) activated not only WT1‑specific CD4+ T cells but also CD8+ T cells that produced IFN‑γ following stimulation with immature dendritic cells (imDCs) pulsed with WT1 killer peptide (imDC/WT1 KP37‑45) in an HLA‑A*02:01‑ or HLA‑A*02:06‑restricted manner. Furthermore, the activated WT1‑reactive CD4+ Th1 cells were predominantly effector memory (EM) T cells. In 5 of 12 (41.7%) patients with cancer carrying the HLA‑A*02:01 or HLA‑A*02:06 allele, WT1‑reactive CD8+ T cells stimulated with mDC/WT1 HP34‑51 enhanced their levels of WT1 KP37‑45‑specific IFN‑γ production, with an increase >10%. Simultaneous activation of CD4+ and CD8+ T cells occurred more often when stimulation with mDC/WT1 HP34‑51 was combined with imDC/WT1 KP37‑45 restimulation. These results indicated that the novel mDC/WT1 HP34‑51 combination induced responses by WT1‑specific EM CD4+ Th1 cells and HLA‑A*02:01‑ or HLA‑A*02:06‑restricted CD8+ CTLs, suggesting its potential as a WT1‑targeting cancer vaccine.

Background: Given improvements in multimodality therapy, survival among children with Wilms tumor (WT) exceeds 90%. However, 15% of children with favorable histology and 50% of children with anaplastic WT experience recurrence or progression. Of patients with advanced disease, only 50% survive to adulthood. In adult malignancies (including renal tumors), patient survival has improved with the advent of immunotherapy. However, little is known about the immune microenvironment of WT, making the potential role of immunotherapy unclear., Objective: The objective of the study is to perform an exploratory, descriptive analysis of the immune milieu in WT., Study Design: Between 2016 and 2017, all pediatric patients with WT, some of whom received neoadjuvant chemotherapy, underwent ex vivo wedge biopsy at the time of nephrectomy. The fresh tumor tissue and peripheral blood samples were analyzed for infiltrating immune infiltrate and effector cells using flow cytometry. Immunohistochemistry was performed for CD4, CD8, and PD-L1 expression. Matched blood samples were obtained for each patient, and circulating immune cells were analyzed by flow cytometry., Results: A total of six patients were enrolled. One patient with neuroblastoma was excluded. The remaining five patients included the following: two with unilateral WT (resected before chemotherapy), two with bilateral WT (resected after neoadjuvant chemotherapy), and one with Denys-Drash syndrome, end-stage renal disease, and history of WT in the contralateral kidney. Immune analysis showed that WT were infiltrated by immune cells regardless of chemotherapy status. CD8 and CD4 T cells were present in the tumor tissue and exhibited an activated phenotype. Elevated levels of natural killer (NK) cells were observed in the tumors (Figure). Immune checkpoint PD-L1 was also found expressed in one of the tumors stained., Discussion: In this pilot study, it was found that WTs were infiltrated by immune cells (CD45+) both before and after chemotherapy. Elevated levels of NK cells infiltrating the tumor specimens, which were quantitatively increased compared with levels of NK cells circulating in the blood, were noted. T cells, particularly CD4+ and CD8+ T cells, were present in tumor specimens. Tumor-infiltrating CD4 and CD8 T cells displayed an activated phenotype as defined by increased expression of human leukocyte antigen-DR isotype (HLA-DR), programmed cell death protein 1 (PD1), and CD57. Together, these findings suggest that WT microenvironment is immune engaged and may be susceptible to immunotherapy similar to other malignancies., Conclusions: These pilot data suggest an immune-engaged tumor microenvironment is present within WT. This implies that WT may be susceptible to immunotherapy similar to adult renal tumors and other adult malignancies. Follow-up studies are currently underway., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Wilms tumor 1 (WT1) oncoprotein is an intracellular oncogenic transcription factor which is barely expressed in normal adult tissues but over expressed in a variety of leukemias and solid cancers. WT1-derived HLA-A*02:01 T cell epitope, RMFPNAPYL (RMF), is a validated target for T cell-based immunotherapy. We generated two T cell receptor mimic antibody-drug conjugates (TCRm-ADCs), ESK-MMAE, and Q2L-MMAE, against WT1 RMF/HLA-A*02:01 complex with distinct affinities, which mediate specific antitumor activity. Although ESK-MMAE showed higher tumor growth inhibition ratio in vivo, the efficacy of them was not so promising, which might be due to low expression of peptide/HLA targets. Therefore, we explored a bispecific TCRm-ADC that exerted more potent tumor cytotoxicity compared with TCRm-ADCs. Hence, our findings validate the feasibility of the presenting intracellular peptides as the targets of ADCs, which broadens the antigen selection range of antibody-based drugs and provides new strategies for precision medicine in tumor therapy.

We recently demonstrated that the major effector function of neonatal CD4 + T cells is to produce CXCL8, a prototypic cytokine of innate immune cells. In this article, we show that CXCL8 expression, prior to proliferation, is common in newly arising T cells (so-called "recent thymic emigrants") in adults, as well as in babies. This effector potential is acquired in the human thymus, prior to TCR signaling, but rather than describing end-stage differentiation, such cells, whether isolated from neonates or adults, can further differentiate into IFN-γ-producing CD4 + T cells. Thus, the temporal transition of host defense from innate to adaptive immunity is unexpectedly mirrored at the cellular level by the capacity of human innate-like CXCL8-producing CD4 + T cells to transition directly into Th1 cells., (Copyright © 2017 The Authors.)

Pancreatic ductal adenocarcinoma (PDA) is characterized by a very poor prognosis, despite novel chemotherapeutic treatments. Moreover, the majority of PDA patients with complete surgical resection show recurrence within 5 years of resection. Therefore, new targeted cancer vaccines are urgently needed to extend PDA patient survival. The Wilms' tumor 1 (WT1) antigen was identified as an excellent antigen in a list of 75 tumor-associated antigens by a National Cancer Institute prioritization project based on several factors, such as therapeutic function. WT1-targeted cancer vaccines are not only efficient in the regression of PDA cells but also angiogenesis and immune suppressive cells, such as myeloid-derived suppressor cells (MDSCs), in the PDA microenvironment. Moreover, WT1 is increased in PDA cells; thus, WT1 may represent an effective therapeutic target for PDA, resulting in a survival benefit for PDA patients. A novel WT1 peptide with increased immunogenicity was developed and used in clinical trials to induce more successful clinical results. This review summarizes the clinical trials of PDA patients receiving WT1-targeted cancer vaccines.

Wilms' tumor 1 (WT1) expression has been investigated in various human cancers as a target molecule for cancer immunotherapy. However, few studies have focused on WT1 expression in dogs. Firstly, cDNA of canine WT1 (cWT1) was molecularly cloned from normal canine kidney. The cross-reactivity of the anti-human WT1 monoclonal antibody (6F-H2) with cWT1 was confirmed via Western blotting using cells overexpressing cWT1. Immunohistochemical staining revealed that cWT1 expression was detected in all canine lymphoma tissues and in some normal canine tissues, including the kidney and lymph node. cWT1 is a potential immunotherapy target against canine cancers.

Wilms' tumors (WT), which accountfor 6% of all childhood cancers, arise from dysregulated differentiation of nephrogenic progenitor cells from embryonic kidneys. Though there is an improvement in the prognosis of WT, still 10% of patients with WT die due to recurrence. Thus more effective treatment approaches are necessary. We previously characterized the inflammatory microenvironment in human WT and observed the robust expression of COX-2. The aim of this study was to extend our studies to analyze the role of COX-2 pathway components in WT progression using a mouse model of WT. Herein, COX-2 pathway components such as COX-2, HIF1-α, p-ERK1/2, and p-STAT3 were upregulated in mouse and human tumor tissues. In our RPPA analysis, COX-2 was up-regulated in M15 cells after Wt1 gene was knocked down. Flow cytometry analysis showed the increased infiltration of immune suppressive inflammatory cells such as pDC's and Treg cells in tumors. The chemotactic chemokines responsible for the infiltration of these cells were also induced in CCR5 and CXCR4 dependent manner respectively. The immunosuppressive cytokines IL-10, TGF-β, and TNF-α were also up-regulated. Furthermore, more pronounced Th2 and Treg induced cytokine response was observed than Th1 response in tumors. Basing on all these evidences it is speculated that COX-2 pathway may be a beneficial target for the treatment of WT. It may be most effective as an adjuvant therapy together with other inhibitors. Thus, our current study provides a good rationale for initiating animal studies to confirm the efficacy of COX-2 inhibitors in decreasing tumor cell growth in vivo., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To study the changes and clinical significance of CD4 + CD25 + CD127 low regulatory T cells (Treg) and CD3 + CD16 + CD56 + natural killer T cells (NKT) in peripheral blood of children with Wilms tumor., Methods: Twenty-one children with Wilms tumor were enrolled as the case group, and twenty-one healthy children for physical examinations were enrolled as the control group. Flow cytometry was used to detect the levels of CD4 + CD25 + CD127 low T cells and CD3 + CD16 + CD56 + T cells in peripheral blood of two groups., Results: The level of Treg cells in peripheral blood of the case group was significantly lower than in the control group (p<0.05). The level of NKT cells in peripheral blood of the case group was significantly higher than in the control group (p<0.05)., Conclusions: Treg cells and NKT cells play important roles in the occurrence and development of Wilms tumor. Treg cells and NKT cells may be useful indexes for evaluating immunological function in children with Wilms tumor.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy.

Secondary hemophagocytic lymphohistiocytosis (HLH) is most commonly associated with malignancy, infection, or an underlying autoimmune disorder. Malignancy-associated hemophagocytic syndrome is responsible for most secondary HLH cases, but it has not been well described in children. We present a case of a 4-year-old female with favorable histology of Wilms tumor who developed secondary HLH after unsuccessful resection of the tumor and initiation of chemotherapy.

Despite Wilms tumor 1 (WT1) protein was originally considered as a specific immunomarker of Wilms tumor, with the increasing use of immunohistochemistry, there is evidence that other tumors may share WT1 protein expression. This review focuses on the immunohistochemical profile of WT1 protein in the most common malignant tumors of children and adolescents. The variable expression and distribution patterns (nuclear vs cytoplasmic) in the different tumors, dependent on the antibodies used (anti-C or N-terminus WT1 protein), will be emphasized by providing explicative illustrations. Potential diagnostic pitfalls from unexpected WT1 protein expression in some tumors will be discussed in order to avoid diagnostic errors, especially when dealing with small biopsies., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Wilms tumor (WT) is a pediatric tumor of the kidney, the treatment of which includes heavy chemotherapy. Affected children would likely benefit from more targeted therapies with limited side effects. Establishment of relevant orthotopic WT xenografts is important to better understand mechanisms of WT growth and for preclinical drug testing., Procedure: Here we established and characterized orthotopic xenografts from WT cell lines WiT49, CCG-99-11, and WT-CLS1 to ascertain in what aspects each of them recapitulated WT histology, immunophenotype, invasion, and metastatic spread., Results: WiT49 xenografts recapitulated near triphasic WTs with clear WT1 staining and anaplastic features, but with tumor restricted to the kidney. On the contrary both CCG-99-11 and WT-CLS1 xenografts conveyed metastatic disease. CCG-99-11 showed a blastemal phenotype whereas WT-CLS1 xenografts did not properly reflect any specific WT subtype., Conclusions: From the three tested cell lines, orthotopic WiT49 xenografts best reflect the triphasic pattern of classical WT., (© 2014 Wiley Periodicals, Inc.)

The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)-A0201 epitope, is a validated target for T cell-based immunotherapy. Using phage display technology, we discovered a fully human "T cell receptor-like" monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1- and HLA-A0201-restricted manner, with high avidity [dissociation constant (Kd)=0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome-positive leukemia in nonobese diabetic/severe combined immunodeficient γc-/- (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.

Wilms' tumor gene WT1 encodes a transcription factor and functions as an oncogene. WT1 gene product WT1 protein is a promising par-tumor-associated antigen. WT1 peptide-based immunotherapy has been performing for more than six hundred patients with leukemias and various types of solid tumors. This immunotherapy is safe and has clinical benefit especially for leukemia, glioblastoma multiforme, advanced pancreatic cancer, and ovarian cancer. As a new strategy for cancer treatment, it should be recommended to initiate immunotherapy that had a potential of eradication of cancer stem cells before surgery, chemo- and radio-therapy.

Wilms Tumor (WT) is the most common renal childhood tumor. Recently, we reported a cDNA microarray expression pattern that varied between WTs with different risk histology. Since the Societé Internationale d'Oncologie Pédiatrique (SIOP) in Europe initiates treatment without a histological confirmation, it is important to identify blood-born markers that indicate WT development. In a multicenter study, we established an autoantibody signature by using an array with 1,827 recombinant E. coli clones. This array was screened with sera of patients with WT recruited by SIOP or the Children's Oncology Group (COG). We report an extended number of antigens that are reactive with autoantibodies present in sera from patients with WT. We established an autoantibody signature that separates untreated patients with WT recruited in SIOP from non-WT controls with a specificity of 0.83 and a sensitivity of 0.82 at standard deviations of 0.02 and 0.04, respectively. Likewise, patients recruited in the COG in the United States were separated from the controls with an accuracy of 0.83 at a standard deviation of 0.02. Proteins that were most significant include zinc finger proteins (e.g., ZFP 346), ribosomal proteins and the protein fascin that has been associated with various types of cancer including renal cell carcinoma. Our study provides first evidence for autoantibody signatures for WTs and suggests that these may be most informative before chemotherapy. We present the first multicenter study of autoantibody signatures in patients with WT. We established an autoantibody signature that separates patients with WT from controls., (Copyright © 2011 UICC.)

Protein fusion to ubiquitin results in its targeting to proteasome and processing through MHC class I pathway. We used this approach to induce cytotoxic T lymphocyte (CTL) response against a MHC class I epitope. Therefore, two known proteasome targeting systems, "ubiquitin fusion degradation" (UFD) and "N-end rule", were used to immunise C57BL/6 mice. Two plasmids encoding an epitope from Wilms' Tumour 1 (WT1-126), fused N-terminally to ubiquitin, were constructed. They were designated as "pUbVVPT" and "pUbGRPT", targeting the fused epitope to UFD and N-end pathways, respectively. A plasmid encoding WT1-126 without ubiquitin fusion (pPT) was also constructed as control. Three mice groups were immunised using these constructs (UGR, UVV and PT groups). Two other groups received mixed immunisations of pUbVVPT or pUbGRPT plus pPT plasmids (UVV+PT and UGR+PT). All mice received a WT1-126 peptide booster. Lymphoproliferative responses following stimulation with WT1-126 were observed in all immunisation groups, with mice receiving the mixture of plasmids eliciting the highest proliferation (UVV+PT>UGR+PT>PT). Moreover, In vivo cytotoxicity assay results revealed highest specific lysis of target cells in UVV+PT group. Tumour growth was decreased in all immunised groups, and was completely abrogated in UGR+PT group. In addition, T(H)1 type cytokines patterns were detected from all immunised groups and WT1-126-specific IFNγ producing lymphocytes were developed in them. These results suggest that the delivery of ubiquitin-fused epitopes along with epitopes alone can be used to optimise the effect of DNA vaccines on the induction of anti-tumour immunity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To induce and activate tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) for cancer immunity, it is important not only to select potent CTL epitopes but also to combine them with appropriate immunopotentiating agents. Here we investigated whether tumor immunity induced by WT1 peptide vaccination could be enhanced by IFN-β. For the experimental group, C57BL/6 mice were twice pre-treated with WT1 peptide vaccine, implanted with WT1-expressing C1498 cells, and treated four times with WT1 peptide vaccine at one-week intervals. During the vaccination period, IFN-β was injected three times a week. Mice in control groups were treated with WT1 peptide alone, IFN-β alone, or PBS alone. The mice in the experimental group rejected tumor cells and survived significantly longer than mice in the control groups. The overall survival on day 75 was 40% for the mice treated with WT1 peptide+IFN-β, while it was 7, 7, and 0% for those treated with WT1 peptide alone, IFN-β alone or PBS alone, respectively. Induction of WT1-specific CTLs and enhancement of NK activity were detected in splenocytes from mice in the experimental group. Furthermore, administration of IFN-β enhanced expression of MHC class I molecules on the implanted tumor cells. In conclusion, our results showed that co-administration of WT1 peptide+IFN-β enhanced tumor immunity mainly through the induction of WT1-specific CTLs, enhancement of NK activity, and promotion of MHC class I expression on the tumor cells. WT1 peptide vaccination combined with IFN-β administration can thus be expected to enhance the clinical efficacy of WT1 immunotherapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.

Wilms' tumor (WT), the most frequent renal solid tumor in children, has been linked to aberrant Wnt signaling. Herein, we demonstrate that different WTs can be grouped according to either sensitivity or resistance to an antibody (Ab) specific to frizzled7 (FZD7), a Wnt receptor. In the FZD7-sensitive WT phenotype, the Ab induced cell death of the FZD7(+) fraction, which in turn depleted primary WT cultures of their clonogenic and sphere-forming cells and decreased in vivo proliferation and survival on xenografting to the chick chorio-allantoic-membrane. In contrast, FZD7-resistant WT in which no cell death was induced showed a different intra-cellular route of the Ab-FZD7 complex compared with sensitive tumors and accumulation of β-catenin. This coincided with a low sFRP1 and DKK1 (Wnt inhibitors) expression pattern, restored epigenetically with de-methylating agents, and lack of β-catenin or WTX mutations. The addition of exogenous DKK1 and sFRP1 to the tumor cells enabled the sensitization of FZD7-resistant WT to the FZD7 Ab. Finally, although extremely difficult to achieve because of dynamic cellular localization of FZD7, sorting of FZD7(+) cells from resistant WT, showed them to be highly clonogenic/proliferative, overexpressing WT 'stemness' genes, emphasizing the importance of targeting this fraction. FZD7 Ab therapy alone or in combination with Wnt pathway antagonists may have a significant role in the treatment of WT via targeting of a tumor progenitor population.

Several studies report autoantibody signatures in cancer. The majority of these studies analyzed adult tumors and compared the seroreactivity pattern of tumor patients with the pattern in healthy controls. Here, we compared the autoimmune response in patients with neuroblastoma and patients with Wilms tumor representing two different childhood tumors. We were able to differentiate untreated neuroblastoma patients from untreated Wilms tumor patients with an accuracy of 86.8%, a sensitivity of 87.0% and a specificity of 86.7%. The separation of treated neuroblastoma patients from treated Wilms tumor patients' yielded comparable results with an accuracy of 83.8%. We furthermore identified the antigens that contribute most to the differentiation between both tumor types. The analysis of these antigens revealed that neuroblastoma was considerably more immunogenic than Wilms tumor. The reported antigens have not been found to be relevant for comparative analyses between other tumors and controls. In summary, neuroblastoma appears as a highly immunogenic tumor as demonstrated by the extended number of antigens that separate this tumor from Wilms tumor.

Background: Efficacy of cancer vaccines may be limited due to immune escape mechanisms like loss or mutation of target antigens. Here, we analyzed 10 HLA-A2 positive patients with acute myeloid leukemia (AML) for loss or mutations of the WT1 epitope or epitope flanking sequences that may abolish proper T cell recognition or epitope presentation., Methods: All patients had been enrolled in a WT1 peptide phase II vaccination trial (NCT00153582) and ultimately progressed despite induction of a WT1 specific T cell response. Blood and bone marrow samples prior to vaccination and during progression were analyzed for mRNA expression level of WT1. Base exchanges within the epitope sequence or flanking regions (10 amino acids N- and C-terminal of the epitope) were assessed with melting point analysis and sequencing. HLA class I expression and WT1 protein expression was analyzed by flow cytometry., Results: Only in one patient, downregulation of WT1 mRNA by 1 log and loss of WT1 detection on protein level at time of disease progression was observed. No mutation leading to a base exchange within the epitope sequence or epitope flanking sequences could be detected in any patient. Further, no loss of HLA class I expression on leukemic blasts was observed., Conclusion: Defects in antigen presentation caused by loss or mutation of WT1 or downregulation of HLA molecules are not the major basis for escape from the immune response induced by WT1 peptide vaccination.

Background: The Wilms' tumor antigen (WT1) is an attractive target for immunotherapy of leukemia. In the past, we isolated and characterized the specificity and function of a WT1-specific T-cell receptor. The goal of this translational study was to develop a safe and efficient WT1-T-cell receptor retroviral vector for an adoptive immunotherapy trial with engineered T cells., Design and Methods: We generated a panel of retroviral constructs containing unmodified or codon-optimized WT1-T-cell receptor alpha and beta genes, linked via internal ribosome entry sites or 2A sequences, with or without an additional inter-chain disulfide bond in the T-cell receptor constant domains. These constructs were functionally analyzed in vitro, and the best one was tested in an autologous primary leukemia model in vivo., Results: We identified a WT1-T-cell receptor construct that showed optimal tetramer staining, antigen-specific cytokine production and killing activity when introduced into primary human T cells. Fresh CD34(+) cells purified from a patient with leukemia were engrafted into NOD/SCID mice, followed by adoptive immunotherapy with patient's autologous T cells transduced with the WT1-T-cell receptor. This therapeutic treatment evidently decreased leukemia engraftment in mice and resulted in a substantial improvement of leukemia-free survival., Conclusions: This is the first report that patient's T cells, engineered to express the WT1-T-cell receptor, can eliminate autologous leukemia progenitor cells in an in vivo model. This study provides a firm basis for the planned WT1-T-cell receptor gene therapy trial in leukemia patients.

The aim of this study was to investigate the frequency of blood count, lymphocyte subpopulations, and immunoglobulin levels alterations in a group of healthy nephroblastoma long-term survivors. The group included 122 nephroblastoma longterm survivors who were at least five years post anticancer therapy and free of any sign of recurrence The proportion of lymphocyte subpopulations was analyzed by flow cytometry using antibodies anti CD45 FITC/CD14 PE, anti CD3 FITC/ CD16+CD56 PE, anti CD4 FITC/ CD8 PE and anti CD20 FITC. Immunoglobulin G, A, and M levels were evaluated by immunoturbidimetry. Total blood count was also examined. The occurrence of decreased immunoglobulin levels, leukocytes, lymphocytes, and granulocytes count, proportion of T lymphocytes and their CD4+ subpopulation are not frequent. The most frequently decreased lymphocyte subpopulation was CD8 (15.5%). The most frequent abnormal findings were increased proportion of NK cells (38.5 %), B lymphocytes (38,52 %), decreased number of erythrocytes (25.2 %), hemoglobin levels (41.7 %) and hematocrit (13.9 %). The only significant differences between results of immunological examination and course of the disease were more frequently decreased proportion of CD4+ lymphocytes in recurrent disease survivors and lower IgA levels in survivors after radiotherapy. We found decreased at least one immunological parameter in one fifth of the survivors. The most frequently altered parameter was hemoglobin, which was decreased in 41.7 % of survivors. Decraesed hemoglobin may worsen quality of survivors life. Key words: nephroblastoma long-term survivors, blood count, lymphocyte subpopulations, immunoglobulin G, A, M serum levels.

The development of cancer vaccines directed against myeloid leukaemias has been a research area of intense interest in the past decade. Both human studies in vitro and mouse models in vivo have demonstrated that leukaemia-associated antigens (LAAs), such as the fusion protein BCR-ABL, Wilms' tumour protein and proteinase 3, may serve as effective targets for cellular immunotherapy. Peptide-based vaccines are able to induce cytotoxic T-lymphocyte responses that kill leukaemia cells. Based on these results, pilot clinical trials have been initiated in chronic and acute myeloid leukaemia and other haematological malignancies, which include vaccination of patients with synthetic peptides derived from these LAAs. Results from these trials show that peptide vaccines are able to induce immune responses that are sometimes associated with clinical benefit. These early clinical results are promising and provide valuable information for future improvement of the vaccines. This chapter will focus mainly on discussing the preclinical studies of peptide vaccines in human systems, the results from clinical trials and the future prospects for vaccine therapy for myeloid leukaemia.

Compelling evidences indicate a key role for regulatory T cells (T(reg)) on the host response to cancer. The Wilms' tumor antigen (WT1) is overexpressed in several human leukemias and thus considered as promising target for development of leukemia vaccine. However, recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of T(regs). We have generated T-cell lines and clones that specifically recognized a WT1-84 (RYFKLSHLQMHSRKH) peptide in an HLA-DRB1*0402-restricted manner. Importantly, they recognized HLA-DRB1*04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a T helper 2 cytokine profile, had a CD4(+)CD25(+)Foxp3(+)GITR(+)CD127(-) T(reg) phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell contact. Priming of alloreactive T cells in the presence of T(regs) strongly inhibited the expansion of natural killer (NK), NK T, and CD8(+) T cells and had an inhibitory effect on NK/NK T cytotoxic activity but not on CD8(+) T cells. Furthermore, priming of T cells with the WT1-126 HLA-A0201-restricted peptide in the presence of T(regs) strongly inhibited the induction of anti-WT1-126 CD8(+) CTL responses as evidenced by both very low cytotoxic activity and IFN-gamma production. Moreover, these T(reg) clones specifically produced granzyme B and selectively induced apoptosis in WT1-84-pulsed autologous antigen-presenting cells but not in apoptotic-resistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 interleukin-5(+)/granzyme B(+)/Foxp3(+) CD4(+) T(regs) in five of eight HLA-DR4(+) acute myeloid leukemia patients. Collectively, our in vitro and in vivo findings strongly suggest important implications for the clinical manipulation of T(regs) in cancer patients.

Two paediatric cancer patients were found to have influenza shortly after receiving chemotherapy. Both presented with neutropenic fever. The first patient, a 15-year-old boy with Hodgkin's lymphoma, recovered without complications. The second patient, a 15-month-old-girl with metastatic Wilms' tumour, died due to severe infectious complications. These cases illustrate that common viruses, such as the influenza virus, can cause fulminant secondary infections in immunocompromised patients. Viruses, including the influenza virus, should always be considered as pathogens in patients with neutropenic fever, and influenza vaccination should be considered in these high-risk patients.

The expression of Wilms' tumor protein (WT1)-derived peptides on malignant cell surfaces and recognition of those peptides by cellular and humoral immune responses suggest that WT1 may be a promising potential target antigen in immunotherapeutic trials. With a high frequency of expression in hematopoietic as well as solid tumors, WT1 is a broadly applicable target. Both in vivo mouse model and in vitro human studies have demonstrated the ability of WT1-specific cytotoxic T-lymphocytes to lyse WT1-expressing malignancies without harming normal tissue. WT1-peptide vaccination, in combination with adjuvants, has demonstrated the ability to activate WT1-specific immune responses and evidence of clinical activity. Because peptide-based vaccines are human leukocyte antigen-restricted, other more broadly applicable strategies are now being developed to activate WT1-specific immune responses, including the use of WT1-specific viral vectors.

Background & Objective: Natural killer (NK) cells have strong potential of killing tumor cells, but it is difficult to expand sufficient NK cells to satisfy the treatment of tumor. This study was to explore the feasibility of efficiently expand NK cells through co-culturing peripheral blood mononuclear cells (PBMCs) and Wilms tumor cell line HFWT., Methods: PBMCs and HFWT cells were co-cultured to expand NK cells. The cytotoxicity of NK cells was measured using 51Cr release assay and crystal violet (CV) staining. The proportions of CD3(+), CD4(+), CD8(+), CD16(+), and CD56(+) cells were detected using flow cytometry., Results: When PBMCs were co-cultured with irradiated HFWT cells for 10-21 days, NK cells were selectively expanded; the proportion of CD56(+)/CD16(+) cells was over 50%. The NK cells killed more than 80% of fresh HFWT cells at an effector/target ratio of 2. The expansion of NK cells required continuous stimulation of PBMCs by HFWT cells., Conclusion: HFWT cells can effectively stimulating PBMCs to sensitively expand human NK cells for adoptive immunotherapy of human tumors.

The Wilms' tumor gene WT1 is overexpressed in leukemias and various types of solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for immunotherapy against these malignancies. Here, we report the outcome of a phase I clinical study of WT1 peptide-based immunotherapy for patients with breast or lung cancer, myelodysplastic syndrome, or acute myeloid leukemia. Patients were intradermally injected with an HLA-A*2402-restricted, natural, or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0, or 3.0 mg per body at 2-week intervals, with toxicity and clinical and immunological responses as the principal endpoints. Twenty-six patients received one or more WT1 vaccinations, and 18 of the 26 patients completed WT1 vaccination protocol with three or more injections of WT1 peptides. Toxicity consisted only of local erythema at the WT1 vaccine injection sites in patients with breast or lung cancer or acute myeloid leukemia with adequate normal hematopoiesis, whereas severe leukocytopenia occurred in patients with myelodysplastic syndrome with abnormal hematopoiesis derived from WT1-expressing, transformed hematopoietic stem cells. Twelve of the 20 patients for whom the efficacy of WT1 vaccination could be assessed showed clinical responses such as reduction in leukemic blast cells or tumor sizes and/or tumor markers. A clear correlation was observed between an increase in the frequencies of WT1-specific cytotoxic T lymphocytes after WT1 vaccination and clinical responses. It was therefore demonstrated that WT1 vaccination could induce WT1-specific cytotoxic T lymphocytes and result in cancer regression without damage to normal tissues.

An anchorage-dependent Wilms' tumor cell line, HFWT, has been found to be extremely susceptible to human natural killer (NK) cells. Here we established a transfectant of HFWT with the green fluorescence protein (GFP) gene, designated GHINK-1 cells, to apply to the activated NK cytotoxicity assay without radioisotope labeling. After being co-cultured with CD3 CD56+ NK cells, GHINK-1 cells released GFP into the medium. The intensity of the fluorescence from the released GFP correlated almost exactly with the radioactivity of a standard 51Cr-release assay performed with suspension-cultured K562 cells. Because it does not require separation of the remaining live target cells by centrifugation, the non-radioisotopic GFP release assay with GHINK-1 cells is a convenient alternative for monitoring human activated NK killing activity.

Mycobacterium aurum was cultured from the Broviac catheter of a 5-year-old child with metastatic Wilms tumor. Removal of the catheter resulted in prompt resolution of the fever and sterilization of the blood culture. This rapidly growing mycobacterium, previously believed to be a commensal, can cause disease in the immunocompromised host.

Purpose: Previous studies have suggested that increased p53 expression is associated with advanced stage and biologically aggressive (chemotherapy resistant) Wilms tumors. We decided to test the hypothesis that increased immunopositivity of p53 is associated with biological aggressiveness in patients with histologically favorable Wilms tumors., Materials and Methods: We reviewed the charts of all patients with unilateral Wilms tumor treated at our institution between 1976 and 2001. Histological characteristics, tumor stage, clinical course and p53 expression as determined by immunohistochemical analysis were determined. All immunohistological evaluations were performed on tissue obtained before administration of chemotherapy., Results: A total of 63 cases of unilateral histologically favorable Wilms tumor were assessed. Five cases (8%) were p53 positive. No significant relationship to p53 expression or stage at presentation was noted in 1 of 21 (5%) stage 1, 3 of 21 (14%) stage 2, 1 of 11 (9%) stage 3 and 0 of 10 stage 4 tumors positive for up-regulation of p53. Of the 5 patients with up-regulated p53 expression 1 (20%) had documented disease progression or relapse while on standard National Wilms Tumor Study chemotherapy. Of the 58 patients who were p53 negative 10 (17%) had disease progression or relapse while on standard National Wilms Tumor Study chemotherapy (p >0.3)., Conclusion: In contrast to previously published studies, we found no correlation of p53 expression to either tumor stage at presentation (p >0.3) or prognosis (p >0.3) in individuals with histologically favorable Wilms tumor assessed for immunopositivity before administration of chemotherapy.

Aims: To evaluate the prognostic value of index Proliferating Cell Nuclear Antigen (PCNA) in Wilms' tumour in children., Methods: The study comprised 64 children aged from 2 days to 13 years treated according to the SIOP (Society International of Oncology Paediatric) and accepted by the PPGGL (Polish Paediatric Group for the Treatment of Solid Tumours). The studies were conducted on tumour tissue removed during surgery, fixed in formalin and embedded in paraffin blocks. Sections (4 microns) were evaluated by immunohistochemistry, using the peroxidase method to determine the expression of PCNA in Wilms' tumour cells by primary monoclonal antibody NCL-PCNA from Novocastra., Results: The percentage of immunopositive cells in particular fragments of the tumour ranged from 0--93%, mean 30.5%, median 25.5%. Mean and median values enabled division of children into two groups: Group A, where the percentage of cells staining with anti-PCNA was <30% and Group B, where this percentage was >30%. The expression of PCNA was evaluated in various stages of advancement, various histological types and depending on the course of disease. The studies revealed the correlation between index PCNA and stage of advancement P<0.01, index PCNA and histological type of Wilms' tumour P<0.025. Moreover we observed that deaths were found more frequently in tumours with index PCNA >30%, P<0.001., Conclusions: PCNA is a useful prognostic factor in Wilms' tumour in children., (Copyright Harcourt Publishers Limited.)

The Thomsen-Friedenreich glycotope (TF) is considered a general carcinoma autoantigen and is therefore of importance in cancer diagnosis and immunotherapy. We report the distribution of the TF glycotope in developing and adult human kidney and renal neoplasms. A monoclonal antibody and the lectin amaranthin were used to study the TF and its sialylated, masked form by immunohistochemistry and immunoblotting. In developing kidney, the TF was restricted to the loop of Henle, distal tubules, and peripheral collecting ducts, whereas its sialylated form was detectable in all epithelial differentiations derived from the 2 embryonic anlagen, the metanephrogenic blastema being unreactive. This pattern was essentially preserved in adult kidney, with TF labeling beginning in the thick ascending limb and extending into the collecting ducts of outer medulla. The sialylated TF glycotope was additionally observed in ascending thin limbs. The TF was exclusively expressed in the luminal cell surface and hence was inaccessible to immune reactions. Analysis of a spectrum of renal neoplasms failed to detect the TF, with the exception of occasional staining of tubules in nephroblastoma. Moreover, the sialylated TF was only detectable in oncocytoma, chromophobe renal cell carcinoma, cystic nephroma, nephroblastoma, and nephroblastomatosis complex and occasionally in type 1 papillary renal cell carcinoma. Thus, the TF and its sialylated form are expressed in normal developing and adult kidney. However, the TF does not seem to represent a tumor-associated glycotope in human kidney, nor does it appear to be of value in diagnosis and immunotherapy of renal neoplasms.

CD31 has been shown to be a sensitive and specific marker for endothelial differentiation among epithelioid and spindled-pleomorphic human neoplasms. However, the role of this marker in the evaluation of small round cell tumors has not been evaluated. Formalin-fixed, paraffin-embedded tissue sections from 276 small round cell tumors, including 85 Ewing's sarcoma/primitive neuroectodermal tumors (ES/PNET), 52 rhabdomyosarcomas, 10 extraabdominal polyphenotypic small cell tumors, six desmoplastic small cell tumors, 11 neuroblastomas, 23 Wilms' tumors, 20 retinoblastomas, 13 esthesioneuroblastomas, and 56 small cell malignant lymphomas were stained with CD31 (JC/70A, 1:40), using a modified avidinbiotin-peroxidase complex technique, after citrate buffer microwave epitope retrieval. Among nonlymphoid small round cell tumors, four of 85 ES/PNET were at least focally reactive. No other lesion in this group was positive. In contrast, the majority of well-differentiated (11 of 17), intermediately differentiated (two of three), and lymphoblastic lymphomas (three of three) were positive. Small cleaved lymphomas (three of 13 follicular, one of 13 diffuse) were less often reactive, whereas small noncleaved lesions were negative. Although reactivity for CD31 in ES/PNET is uncommon, the presence of platelet/endothelial cell adhesion molecule in a small cell neoplasm should not in isolation be taken as evidence of hematopoietic origin. These results further define the utility of CD31 in the evaluation of human neoplasms.

The Wilms' tumor gene WT1 is expressed at high levels not only in acute myelocytic and lymphocytic leukemia and in chronic myelocytic leukemia but also in various types of solid tumors including lung cancers. To determine whether the WT1 protein can serve as a target Ag for tumor-specific immunity, three 9-mer WT1 peptides (Db126, Db221, and Db235), which contain H-2Db-binding anchor motifs and have a comparatively higher binding affinity for H-2Db molecules, were tested in mice (C57BL/6, H-2Db) for in vivo induction of CTLs directed against these WT1 peptides. Only one peptide, Db126, with the highest binding affinity for H-2Db molecules induced vigorous CTL responses. The CTLs specifically lysed not only Db126-pulsed target cells dependently upon Db126 concentrations but also WT1-expressing tumor cells in an H-2Db-restricted manner. The sensitizing activity to the Db126-specific CTLs was recovered from the cell extract of WT1-expressing tumor cells targeted by the CTLs in the same retention time as that needed for the synthetic Db126 peptide in RP-HPLC, indicating that the Db126-specific CTLs recognize the Db126 peptide to kill WT1-expressing target cells. Furthermore, mice immunized with the Db126 peptide rejected challenges by WT1-expressing tumor cells and survived for a long time with no signs of autoaggression by the CTLs. Thus, the WT1 protein was identified as a novel tumor Ag. Immunotherapy targeting the WT1 protein should find clinical application for various types of human cancers.

Reported here is the clinical presentation and management of patients with rapidly growing non-tuberculous mycobacterial infection diagnosed in a paediatric oncology unit. A retrospective analysis that correlated patient isolates with the children's cancer registry revealed two cases of non-tuberculous mycobacterial infection; both had been observed within the last 6 years and were due to Mycobacterium chelonae. The first case was line-associated and the second was a disseminated infection. In both cases the patients were lymphopenic and had had indwelling vascular catheters. Neither patient was neutropenic. The literature on mycobacterial infection in children with cancer is also reviewed.

The existence of specific immunity against pediatric tumors is not well studied. We report here the CD3+CD4-CD8+ cytotoxic T lymphocyte (CTL) line established from tumor-infiltrating lymphocytes (TIL) of a 3-mo-old child with Wilms' tumor. This CD3+CD4-CD8+ CTL line showed cytotoxicity against the HLA-A2402+ tumor cells including the autologous tumor, adenocarcinomas from various organs (colon, stomach, lung, and ovary), and an esophageal squamous cell carcinoma. No other cell lines examined, including HLA-A2402- tumors, K562 cells, or HLA-A2402+ normal cells were lysed by this CTL line. This CTL line recognized an HLA-A2402- ovarian adenocarcinoma transfected with HLA-A2402 cDNA. These results suggest the existence of HLA-A2402-restricted and tumor-specific CTL at the tumor site of a child with Wilms' tumor.

In several childhood diseases which have the ensuing risk of infertility in adult life because of direct hypothalamic-pituitary-testicular axis involvement, or as a consequence of therapeutic toxicity, the role of antisperm antibodies (ASA) is rarely addressed. The aim of this study was to investigate the occurrence of ASA in a large prepubertal male population (aged 1.2-13 years) consisting of three groups: Group I, 52 patients affected by malignant diseases (lymphoblastic leukaemia, malignant lymphoma, or Wilm's tumour, n = 42), or by nephrotic syndrome (n = 10); Group II, 212 patients with either genital tract abnormalities (cryptorchidism, inguinal hernia, funicular torsion or hypospadias, n = 202), or cystic fibrosis (n = 10); Group III: 100 age-matched normal boys. Group I and II patients were investigated at diagnosis and during or after treatment (drug, radiation or surgical therapy). Group III was used as controls. ASA were detected in sera by the Tray Agglutination Test (TAT) and indirect IgG, IgA and IgM immunobead tests (iIBT). All normal boys were ASA-negative using both tests. Twenty-six out of the 264 patients (9.8%) in Groups I and II were ASA-positive: 23 (8.7%) patients had a positive TAT with a titre of 1:32 to 1:128, whilst 14 (5.3%) had IgG-ASA after iIBT. Eleven patients (4.1%) were ASA-positive in both tests. Of the 26 ASA-positive boys, 24 had genital tract abnormalities (cryptorchidism, testicular torsion, hypospadias) and two had leukaemia with testicular infiltration. Treatment did not modify antibody positivity. Our data confirm that ASA can occur in prepubertal boys, mostly among cases with urogenital pathology, but that it is rare among other cases. Therefore autoimmune reaction against spermatozoa is another factor that should be considered in the evaluation of several conditions in childhood involving reproductive tract alteration and potential impairment of the blood testis (Sertoli cell) barrier.

To provide an objective assessment of the comparative utility of fluorescence- and peroxidase-based immunohistochemistry and electron microscopy, an observer blinded study was conducted under realistic study conditions utilizing a large sampling of poorly differentiated pediatric round cell tumors. Working independently, using a single ancillary technique of particular expertise, each of three investigators attempted to render a specific diagnosis with regard to 50 diagnostically challenging tumors. The results were compared against the subsequent "file diagnosis" established by consensus with all relevant information made available. A grading scheme was applied wherein points were awarded based on the accuracy and confidence of diagnosis. A comparative efficiency rating, expressed as a percentage, was formulated by dividing the number of points awarded each technique by the total number of points theoretically available. Electron microscopy proved superior overall, with an efficiency rating of 89%. Immunoperoxidase and immunofluorescence studies yielded efficiency ratings of 71 and 61%, respectively. Used in combination, the techniques achieved an efficiency rating of 95%. Application of these ancillary techniques resulted in a revision of the provisional diagnosis in 11 of 50 cases, and left only two cases without a firm specific diagnosis.

The anaplastic variant of Wilms' tumor is regarded as the result of tumor progression of the more common classic Wilms' tumor. Anaplasia is rare and occurs in only 4.5% of tumors. Three anaplastic Wilms' tumors in our collection were examined in comparison with 10 classic Wilms' tumors for p53 expression by immunohistochemical techniques and Northern blot analysis, and their p53 gene structure was determined by single-stranded conformation polymorphism and sequence analysis. All classic tumors contained a wild-type p53 gene and expressed marginal levels of protein as expected for normal p53. In contrast, three out of three anaplastic tumors demonstrated evidence of p53 alterations consistent with a role of p53 in tumor progression. One of the anaplastic mutants (W4) did not express protein or p53 mRNA. Its apparently normal immunophenotype would have disguised the mutated nature of p53, which was detected only by mRNA and sequence analysis. The second anaplastic mutant (W16) contained normal levels of p53 mRNA, but overexpressed the protein in a fashion typical of mutated p53. The same immunophenotype was displayed by fixed primary tissue of the third anaplastic tumor (W17), but p53 mutation could not be confirmed for lack of frozen primary material. The present study emphasizes the importance of p53 function in the anaplastic progression of Wilms' tumor and the risk of error in assessing normal p53 function using a single methodology.

A new antigen was detected using a monoclonal antibody generated against malignant blastema from a Wilms' tumor. This antigen showed variable expression in malignant blastemal cells but was never detected in normal blastema of fetal kidneys irrespective of gestational stage. In a series of 16 Wilms' tumors, the most intense and consistent staining was seen in tubule-associated epithelial cells. Such tubular staining is not surprising as the putative induction of malignant blastema to differentiate into malignant tubules is thought to parallel normal tubulogenesis. This antigen was also associated with epithelial cells located in a variety of fetal kidney structures. Again, the staining was most consistent in tubular epithelia. This monoclonal antibody reactive with a blastemal-epithelial-tubular (BET) antigen should be of value in studying the induction of epithelial differentiation in the normal and diseased human kidney.