Maiko Matsushita

Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia

PRAME (Preferentially expressed antigen of melanoma) has been previously identified as a melanoma antigen recognized by cytotoxic T cells (CTLs) and found to be expressed in a variety of cancer cells including leukaemic cells. We have screened 98 Japanese patients with leukaemia and lymphoma for expression of the PRAME gene using semiquantitative reverse transcription polymerase chain reaction (RT‐PCR). Forty‐one patients (42%) showed high levels of PRAME expression. Eight of these patients were then monitored using real‐time PCR for a period of 10–37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the two patients who relapsed, one of which was before cytological diagnosis. These changes were correlated with those of other known genetic markers, such as the bcr‐abl gene. Therefore, quantitative monitoring of the PRAME gene using real‐time PCR method may be useful for detecting minimal residual disease and to predict subsequent relapse, especially in patients without known genetic markers. In addition, a PRAME‐positive leukaemia cell line and fresh leukaemic cells were found to be susceptible to lysis by PRAME‐specific CTLs established from a patient with melanoma, suggesting that the PRAME peptide can also be a target leukaemia antigen for T cells.

Preferentially expressed antigen of melanoma (PRAME) in the development of diagnostic and therapeutic methods for hematological malignancies.

PRAME (Preferentially expressed antigen of melanoma), highly expressed in various solid tumor cells and normal testis, was first isolated as a human melanoma antigen recognized by cytotoxic T cells (CTL). This gene was also expressed in some of the hematological malignancies, including acute myelogenous leukemia (AML) and multiple myeloma. We and others have extensively evaluated the PRAME expression in various hematological malignancies and demonstrated high expression of the PRAME gene in subsets of AML, chronic myelogenous leukemia, acute lymphocytic leukemia, lymphoma and multiple myeloma. In addition, we have demonstrated that PRAME was a useful marker for detection of minimal residual disease (MRD) in patients with leukemia, particularly those leukemias in which tumor specific markers are currently unavailable. Since PRAME was first identified as a tumor antigen recognized by T cells, the possibility that PRAME is a leukemia antigen recognized by T cells was evaluated, and it was found that PRAME-positive leukemia cell lines and fresh leukemia cells were susceptible to lysis by the PRAME-specific CTL. Five CTL epitopes associated with either HLA-A * 0201 or HLA-A * 2402 have recently been identified. It is, therefore, an attractive strategy to apply PRAME specific immunotherapy on patients with PRAME positive leukemia in MRD condition.

Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer

To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high‐grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty‐eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue‐specific expression, KU‐BL‐1 and KU‐BL‐2 were identified as immunogenic antigens with restricted tissue expression. KU‐BL‐1 was found to be a putative human lipoic acid synthetase with a metal‐binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU‐BL‐1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU‐BL‐2 was found to be a putative human kelch‐like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU‐BL‐2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU‐BL‐2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA–A24 binding KU‐BL‐2 peptides. Therefore, KU‐BL‐1 and KU‐BL‐2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.

Possible involvement of allogeneic antigens recognised by donor-derived CD4 cytotoxic T cells in selective GVL effects after stem cell transplantation of patients with haematological malignancy.

Cytotoxic T lymphocyte (CTL) lines specific for allogeneic antigens were generated by in vitro stimulation of donor‐derived peripheral blood mononuclear cells obtained from patients who received human leucocyte antigen (HLA)‐matched allogeneic haematopoietic stem cell transplantation (HSCT). One of the allogeneic antigen‐specific CD4+ CTL lines, CTL‐A, generated from a patient with T cell acute lymphoblastic leukaemia, recognised HLA‐DPB1*0501‐positive Epstein–Barr virus‐immortalised human B cell line (EBV‐B cells), phytohaemagglutinin blasts and leukaemia cells, but not interferon‐γ (IFN‐γ) treated HLA‐DPB1*0501‐positive fibroblasts, indicating that this CD4+ T‐cell line recognised a minor histocompatibility antigen (mHa) that is preferentially expressed in haematopoietic cells in an HLA‐DPB1*0501‐restricted manner. The other CD4+ CTL line, CTL‐B, generated from a patient with chronic myeloid leukaemia, recognised mismatched HLA‐DQB1*0303 on EBV‐B cells and phytohaemagglutinin (PHA) blasts. Interestingly, this CTL line did not recognise IFN‐γ‐treated recipients skin fibroblasts, as HLA‐DQ was merely upregulated even after IFN‐γ stimulation in non‐haematopoietic cells including fibroblasts, endothelial cells and hepatocytes. These results suggest that these CD4 positive CTLs, specific for mismatch HLA‐DQ and mHa that are preferentially expressed on haematopoietic cells, may play an important role in induction of selective graft‐versus‐leukaemia effect without development of graft‐versus‐host disease after allogeneic HSCT.

A Novel Phthalimide Derivative, TC11, Has Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies.

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2271-279). The CTLs induced by PEPP2271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.

Codon 72 polymorphism of TP53 gene is a novel prognostic marker for therapy in multiple myeloma

Despite recent advances in the treatment of multiple myeloma (MM), patients with high-risk chromosomal abnormalities, such as deletion of chromosome 17 (del 17), on which the TP53 tumour suppressor gene is localized, revealed significantly shorter survival. In addition to the dominant effect of TP53 deletion, the association between TP53 polymorphism at codon 72 and cancer risk in various types of cancers has been widely studied. Namely, the transition of the

Synthesis and biological evaluation of the natural product komaroviquinone and related compounds aiming at a potential therapeutic lead compound for high-risk multiple myeloma

Alternatives of treatments for multiple myeloma (MM) have become increasingly available with the advent of new drugs such as proteasome inhibitors, thalidomide derivatives, histone deacetylase inhibitors, and antibody drugs. However, high-risk MM cases that are refractory to novel drugs remain, and further optimization of chemotherapeutics is urgently needed. We had achieved asymmetric total synthesis of komaroviquinone, which is a natural product from the plant Dracocephalum komarovi. Similar to several leading antitumor agents that have been developed from natural compounds, we describe the antitumor activity and cytotoxicity of komaroviquinone and related compounds in bone marrow cells. Our data suggested that komaroviquinone-related agents have potential as starting compounds for anticancer drug development.

Optimal Anti-cancer Drug Profiles for Effective Penetration of the Anti-cancer Drug Market by Generic Drugs in Japan:

Background: The increased use of generic drugs is a good indicator of the need to reduce the increasing costs of prescription drugs. Since there are more expensive drugs compared with other therapeutic areas, “oncology” is an important one for generic drugs. The primary objective of this article was to quantify the extent to which generic drugs in Japan occupy each level of the Anatomical Therapeutic Chemical (ATC) classification system. Methods: The dataset used in this study was created from publicly available information obtained from the IMS Japan Pharmaceutical Market database. Data on the total amount of sales and number of prescriptions for anti-cancer drugs between 2010 and 2016 in Japan were selected. The data were categorized according to the third level of the ATC classification system. Results: All categories of the ATC classification system had increased market shares in Japan between 2010 and 2016. The barriers to market entry were relatively low in L01F (platinum anti-neoplastics), L01C (plant-based neoplastics), L02B (cytostatic hormone antagonists), and L01D (anti-neoplastic antibiotics) but were high in L02A (cytostatic hormones), L01H (protein kinase inhibitors), and L01B (anti-metabolites). Conclusions: Generic cancer drugs could bring savings to Japanese health care systems. Therefore, their development should be directed toward niche markets, such as L02A, L01H, and L01B, and not competitive markets.

Anticancer Drug Prescription Patterns in Japan: Future Directions in Cancer Therapy

Background: Despite their benefits, the rapid development of new cancer treatments has been a significant driver of increasing health care expenditures in the face of limited health care budgets. In this study, we analyzed the prescribing trends for anticancer drugs from 2010 through 2016 in Japan and sought to identify unique trends that could provide a basis for future medical economic research aiming to develop more efficacious and cost-effective cancer therapies. Methods: We used publicly available marketing data for anticancer drugs in Japan for 2010-2016. The drugs selected for this research were categorized according to the Anatomical Therapeutic Chemical Classification System. We investigated the overall anticancer drug market size, the number of anticancer drugs, the top 30 selling anticancer categories, sales and prescription volumes, and changes in sales and prescription volumes between 2010 and 2016 in the country. Results: The anticancer agent market expanded each year from 2010 to 2016, with sales exceeding 1 trillion yen in 2015. The proportion of molecular targeted drugs (antineoplastic mAbs and protein kinase inhibitors) among the top 30 selling anticancer categories has continued to increase, and both the sales and prescription volumes of these drugs exceeded those of drugs in other categories, suggesting that these treatments play a dominant role in cancer pharmacotherapy. Conclusion: The availability and increasing use of innovative but more expensive targeted therapies were major drivers of increases in pharmaceutical expenditures for cancer treatment in Japan. Therefore, the effective use of genetic testing can mitigate these rising costs.