Yumiko Higuchi

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.

CD38 expression by hematopoietic stem cells of newborn and juvenile mice

While investigators in a number of laboratories have documented that the hematopoietic stem cells (HSCs) of fetal and adult mice are CD38+, no information is available about CD38 expression by HSCs of newborn and juvenile mice. We used a murine transplantation model to examine HSC CD38 expression. First, we observed that all HSCs from newborn bone marrow are CD38−. Next, it was determined that the majority of HSCs in the bone marrow of 5-week-old mice are CD38−, with a minority being CD38+. These observations indicated that the CD38+ subpopulation of HSC appears before the age of 5 weeks and expands during adolescence. However, the majority of HSCs of 5-week-old mice became CD38+ following injection of 5-fluorouracil, indicating that activation of juvenile stem cells enhances CD38 expression. These observations may have implications for CD38 expression by HSCs from human umbilical cord blood and bone marrow of young children in steady state and under pathological conditions.

Simple polymerase chain reaction for the detection of mutations and deletions in the epidermal growth factor receptor gene: Applications of this method for the diagnosis of non-small-cell lung cancer

BACKGROUND Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the responses to the tyrosine kinase inhibitors gefitinib and erlotinib in patients with non-small-cell lung cancer (NSCLC). Although various methods for detecting EGFR gene mutations have been developed, they have several disadvantages. We attempted to establish a new method for the detection of EGFR gene mutations with the use of paraffin-embedded samples. METHODS The detections of T790M mutations in exon 20 and L858R mutations in exon 21 are based on the principle of allele-specific oligonucleotide polymerase chain reaction (PCR). We also designed PCR primers that enable to detect all types of deletions in exon 19. We assessed the basic performance efficiency of this method, and to confirm its clinical applicability, we performed PCR using DNA extracted from 66 tissue sections that were obtained from patients with NSCLC and embedded in paraffin. RESULTS The sensitivity of this method for the detection of deletions or mutations was as low as 0.5%. In the 66 subjects whose samples were analyzed, we detected the following deletions and mutations in the EGFR gene: 11 deletions in exon 19, 8 L858R mutations, and 1 double mutation of L858R and T790M. CONCLUSION The present method is sensitive and specific for the detection of deletions and mutations in the EGFR gene and is thus suitable for use in laboratory tests.

Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focusing method

To analyze both hemoglobin (Hb) and globin chain variants, we modified a commonly used method, capillary isoelectric focusing (CIEF), with detection at 280 nm. The samples were hemolysates prepared from red blood cells, and globin chains obtained from the hemolysates by treatment with cold acidified acetone. When the migration time for the internal reference, carbonic anhydrase I (isoelectric point, pI 6.60), was taken as 1.0, the migration ratio for Hb A0 in normal human blood was 0.877 ± 0.004 (mean ± SD, n = 9), and those of the α and β‐ and β‐globin chains were 0.673 ± 0.004 and 0.847 ± 0.005 (mean ± SD, n = 4), respectively. The ratio of peak heights between the β‐ and α‐globin chains (β/α) in the normal Hbs obtained from four subjects was almost constant at 2.5 ± 0.1 (mean ± SD). This ratio indicates which of the globin chains includes a mutation (if one exists). When an Hb variant, Hb Hoshida (in which Gln is substituted for Glu at residue 43 in the β‐globin chain), was analyzed by this method, two main peaks were observed (migration ratios 0.836 and 0.877, corresponding to an abnormal and the normal Hb, respectively). An additional peak with an abnormal migration ratio of 0.788 was also detected in the globin chain profiles. The ratio of peak heights between normal β‐ and α‐globin chains was 1.57, indicating that a mutation exists in the β‐globin chain. We thus established a convenient system using CIEF that provides a rapid and reproducible method for the random analysis of both Hb and globin chain variants.

Dendritic Cell-Based Adjuvant Vaccination Targeting Wilms' Tumor 1 in Patients with Advanced Colorectal Cancer

Despite significant recent advances in the development of immune checkpoint inhibitors, the treatment of advanced colorectal cancer involving metastasis to distant organs remains challenging. We conducted a phase I study to investigate the safety and immunogenicity of Wilms’ tumor (WT1) class I/II peptides-pulsed dendritic cell DC vaccination for patients with advanced colorectal cancer. Standard treatment comprising surgical resection and chemotherapy was followed by one course of seven biweekly administrations of 1–2 × 107 DCs with 1–2 KE of OK-432 (streptococcal preparation) in three patients. Clinical efficacy was confirmed based on WT1 expression using immunohistochemistry on paraffin-embedded tissues and immune monitoring using tetramer analysis and enzyme-linked immunosorbent spot (ELISPOT) assays. WT1 expression with human leukocyte antigen (HLA)-class I molecules was detected in surgical resected tissues. Adverse reactions to DC vaccinations were tolerable under an adjuvant setting. WT1-specific cytotoxic T cells were detected by both modified WT1-peptide/HLA-A*24:02 tetramer analysis and/or interferon-γ-producing cells through the use of ELISPOT assays after the first DC vaccination. Immunity acquired from DC vaccination persisted for two years with prolonged disease-free and overall survival. The present study indicated that DC vaccination targeting WT1 demonstrated the safety and immunogenicity as an adjuvant therapy in patients with resectable advanced colorectal cancer.

Plasminogen Kanagawa‐I, a novel missense mutation, is caused by the amino acid substitution G732R

A new dysplasminogen, plasminogen Kanagawa‐I, was identified in a healthy male with no previous thrombotic episodes. His plasma plasminogen (PLG) activity was 51.4% of that of normal pooled plasma (reference interval 70–130%) and the antigen level was 94.2% of that of normal pooled plasma (reference interval 80–150%). Nucleotide sequencing revealed a heterozygous G to A transition in exon 18, which resulted in an amino acid substitution of G732R. Both the probands father and paternal grandfather were heterozygous for this mutation. Interestingly, the grandfather was found to be a compound heterozygote for plasminogen Kanagawa‐I and Tochigi (A601T), so that his plasminogen activity and antigen level was 7.7% and 87.2% of that of normal pooled plasma, respectively. However, he has never been affected by significant thrombosis.

An update on Dendritic Cell-Based Cancer Immunotherapy

Although treating advanced cancers that affect organs with distant metastasis remains challenging, the pace of recent advances has accelerated; these advances have particularly focused on the inhibitors of key immune potentiates. Research on therapeutic vaccination involving active dendritic cell (DC)-based immunotherapy is also being performed for the induction of an effi cient immune response against cancer-associated antigens by the acquired immune system. Cancer vaccines prepared with autologous monocyte-derived mature DCs have been generated using granulocyte–macrophage colony-stimulating factor and interleukin-4, which are principally attributed to the presence of tumor-associated antigens. Wilms’ tumor 1 (WT1) is an attractive target antigen that is widely detected in many cancers. DC-based immunotherapy targeting WT1 may elicit a strong therapeutic response to cancers. DC vaccines primed with HLA class I/II-restricted WT1 peptides (WT1-DC) are a feasible option for patients with advanced cancers. Immune response monitoring using tetramer analysis and/or enzyme-linked immunosorbent spot assay has been applied to determine the effi cacy of WT1-DC. The inhibition of immune suppressors and acceleration of anti-cancer immunity with WT1-DC may comprise a promising future therapeutic strategy for treating advanced cancers.

Quality Verification of Dendritic Cell-Based Cancer Vaccine

In the era of personalized cancer therapy, immunotherapy is now emerging as a potential option. Therapeutic cancer vaccination has been developed for the induction of an efficient immune response targeting tumor-associated antigens. The efficacy of dendritic cell (DC)-based vaccines is attributed to their ability to induce immunity against cancers. Criteria for the approval of therapeutically active DCs, such as their viability and purity, are here verified based on phenotypic characteristics, linked to their antigen-presenting ability and functional analyses of phagocytosis and pinocytosis. A standardized phenotype of DCs harboring bioactive functions would be useful to provide personalized vaccines for cancer immunotherapy.

Induction of Antigen-Specific Cytotoxic T Lymphocytes by Chemoradiotherapyin Patients Receiving Wilmsâ Tumor 1-Targetted Dendritic Cell Vaccinations forPancreatic Cancer

Despite recent advances in cancer treatment, the prognosis of pancreatic cancer (PC) remains poor. Dendritic cells (DCs) play a central role in acquired immunity; therapeutic DC vaccinations have recently been developed for advanced PC. Here we present two cases of PC: inoperable PC localized to the pancreatic head (Case 1, stage IV) and local recurrence complicated by distant metastases following resection of the pancreas body and tail (Case 2, stage III). Both patients received DC vaccinations pulsed with human leukocyte antigen (HLA)-Class I/II-restricted Wilms’ tumor 1 (WT1) peptides during chemoradiotherapy. The induction of WT1 antigen-specific cytotoxic T cells (WT1-CTL) was markedly increased by chemoradiotherapy and was confirmed by measurement of WT1 tetramers and enzyme-linked immunosorbent spot (ELISpot) in both cases. WT1-CTL was found to persist at 1 year without additional DC vaccines in Case 1. In cases 1 and 2, the overall survival (OS) was 32.1 and 24.7 months, respectively, and progression-free survival (PFS) was 25.2 and 8.7 months, respectively. Adverse reactions due to the DC vaccination were tolerable even during chemoradiotherapy, resulting in disease stability. The findings of the present cases may form treatment strategies involving DC vaccination for PC.

Enzyme-Linked Immunosorbent Spot Assay for the Detection of Wilms’ Tumor 1-Specific T Cells Induced by Dendritic Cell Vaccination

Background: Despite recent advances in cancer immunotherapy and the development of various assays for T cell assessment, a lack of universal standards within immune monitoring remains. The objective of this study was to evaluate the enzyme-linked immunosorbent spot (ELISpot) assay in comparison with major histocompatibility complex-tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy. Methods: The ELISpot assay was performed on peripheral blood mononuclear cells to assess reproducibility, daily precision, and linearity using HLA-A*24:02-restricted Cytomegalovirus peptide. Wilms’ tumor 1 (WT1) antigen-specific cytotoxic T cells were then evaluated by both the ELISpot assay and WT1 tetramer analysis in peripheral blood from 46 cancer patients who received DC vaccinations pulsed with human leukocyte antigen (HLA)-A*24:02-restricted modified WT1 peptides. Results: The ELISpot assay was proven to have reproducibility (coefficient of variation (CV) ranged from 7.4% to 16.3%), daily precision (CV ranged from 5.0% to 17.3%), and linearity (r = 0.96–0.98). WT1-specific immune responses were detected by the ELISpot assay in 34 out of 46 patients (73.9%) post-vaccination. A Spearman’s rank-correlation coefficient of 0.82 between the ELISpot assay and WT1 tetramer analysis was obtained. Conclusion: This is the first report of a comparison of an ELISpot assay and tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy. The ELISpot assay has reproducibility, linearity, and excellent correlation with the WT1 tetramer analysis. These findings suggest that the validated ELISpot assay is useful to monitor the acquired immunity by DC vaccination targeting WT1.