Recent evidence suggests that the targeting of membrane transporters specifically activated in cancer stem cells (CSCs) is an important strategy for cancer therapy. The objectives of the present study were to investigate the ion channel expression profiles in digestive CSCs. Cells strongly expressing CSC markers, such as ALDH1A1 and CD44, were separated from the human esophageal squamous cell carcinoma, gastric cancer, and pancreatic cancer cell lines using fluorescence-activated cell sorting, and CSCs were identified based on tumorsphere formation. Messenger RNA levels of CSC markers were higher in CSCs than in non-CSCs. These CSCs also exhibited resistance to anticancer agents. The microarray analysis revealed that the expression of transient receptor potential vanilloid 2 (TRPV2), voltage-gated calcium channels (VGCCs), and voltage-gated potassium channels (VGKCs) were upregulated in esophageal, gastric, and pancreatic CSCs, respectively, compared with non-CSCs. The TRPV2 inhibitor tranilast, VGCCs inhibitors amlodipine and verapamil, and VGKC inhibitor 4-aminopyridine exhibited greater cytotoxicity in CSCs compared with non-CSCs, and their inhibitory effects were also confirmed in a xenograft model in nude mice. Taking these results, phase I/II study to investigate clinical safety and efficacy of neoadjuvant combination chemotherapy of tranilast in advanced esophageal squamous cell carcinoma (TNAC study) is ongoing. These researches identified a role of ion channels in the persistence of CSCs and suggested that their inhibitors may have potential as a therapeutic agent for digestive cancers.

On March 28th, 2022, asciminib hydrochloride (Scemblix® Tablets 20 ‍mg/40 ‍mg), the world's first tyrosine kinase inhibitor (TKI) specifically targeting the ABL myristoyl pocket (STAMP inhibitor), was approved for chronic myeloid leukemia (CML) resistant or intolerant to prior therapy. Asciminib specifically binds to the myristoyl pocket, an allosteric site of BCR::ABL1, and inhibits the ABL1 family molecules. In vitro and in vivo pharmacology studies demonstrated cell growth inhibition and antitumor effects of asciminib. The international phase I study for patients with chronic or accelerated phase CML investigated the maximum tolerated dose (MTD) and recommended dose for expansion (RDE) of asciminib monotherapy. However, the MTD was not reached, so and RDE was determined based on tolerability, safety, pharmacokinetics (PK) and preliminary efficacy data obtained by the time of the study. RDE was determined to be 40 ‍mg twice daily in chronic or accelerated phase CML without T315I mutation, and 200 ‍mg twice daily in chronic or accelerated phase CML with T315I mutation. The international phase III study for patients with chronic phase CML who were previously treated with ≥2 TKIs and resistant or intolerant to the recent treatment demonstrated the superiority of asciminib over bosutinib in achieving the primary endpoint of a major molecular response (MMR) at week 24. Regarding safety, the most common treatment-related adverse event in asciminib arm was thrombocytopenia, and others included neutropenia. Asciminib is expected to be a new treatment option for CML patients who have limited choices due to resistance or intolerance to previous therapies.

Although several types of chimeric antigen receptor (CAR)-modified T cells (CAR-T cells) targeting myeloid antigens have been developed for acute myeloid leukemia (AML) globally, significant clinical benefits have not yet been reported. Furthermore, CAR-T cells targeting juvenile myelomonocytic leukemia (JMML) have not yet been developed. All JMML cells and 63-83% of AML cells express granulocyte macrophage-colony stimulating factor (GM-CSF) receptor (GMR, CD116/CD131 complex). Therefore, we created ligand-based CAR-T cells targeting GMR using the piggyBac transposon system. We further redesigned the CAR construct by optimizing the affinity of the antigen-binding region and length of the spacer region. The GMR CAR-T cells with a mutated GM-CSF at residue 21 (E21K) and a G4S spacer showed superior antitumor effects in the human AML-xenograft model. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. Based on the promising results of the nonclinical study, we started a first-in-human clinical trial of GMR CAR-T cells in patients with CD116-positive AML and JMML in 2021.

Progress in genomic analysis are expected to improve the treatment outcome of cancers of unknown primary(CUP). We conducted a randomized phase Ⅱ trial of carboplatin plus paclitaxel versus site-specific therapy based on gene expression profiling(GEP)in patients with CUP. A phase Ⅱ trial was conducted in patients with CUP to evaluate the feasibility of site- specific therapy based on NGS-based primary site prediction from GEPs, including irGEP. We proposed the possibility of treatment with immune checkpoint inhibitors for CUP patients. Based on the results, a physician-led clinical trial of nivolumab for CUP was conducted with favorable results.

Neurofibromatosis type 2(NF2)is a hereditary condition that causes bilateral vestibular schwannomas(VS), multiple schwannomas, and meningiomas. The prognosis is poor because the multiplicity of the tumors leads to a progressive decline in the quality of life, deafness, and death in an early age. NF2 is caused by a disorder in the tumor suppressor gene NF2, which encodes the merlin protein. Although it is an autosomal dominant disease, more than half of cases are presumed to be de novo caused by somatic mosaicism, the diagnosis rate of which has been improved by the recently introduced technology of targeted deep sequencing of DNA from multiple tissues. No chemotherapeutic drugs for treating NF2-related VS are available at present, and surgery and radiotherapy remain the only therapeutic options. Recently, a randomized, double-blind, multicenter clinical trial has started in Japan to verify the efficacy and safety of bevacizumab, a humanized monoclonal antibody that targets vascular endothelial growth factor, in treating NF2-related VS.

There has been remarkable progress in systemic therapy for advanced lung cancer in recent years. Novel molecular targeted agents directed against oncogenic driver mutations as well as combination strategies with immune checkpoint inhibitors have been continuously emerged in the clinical practice, which is driving the expansion of precision medicine. As most of these newly developed drugs and therapies were approved on the basis of global randomized studies, the robust data for the efficacy and safety focused on the Japanese patients are limited. Given that the genetic, environmental, and social backgrounds of the Japanese are different from those of Caucasians, it is questionable whether the available global data for the efficacy and safety of newly developed drugs can passively be extrapolated to the Japanese patients. In this article, we discuss the regional and racial differences of the responses or toxicity profiles of anti-cancer agents, and review the characteristics of the Japanese subgroup data in global clinical studies focusing on the following 3 different types of drugs: epidermal growth factor receptor-tyrosine kinase inhibitors, cytotoxic chemotherapeutic agents, and immune checkpoint inhibitors.

We analyzed the prognostic significance of chromosomal findings in children with acute lymphoblastic leukemia (ALL), treated according to the Children's Cancer and Leukemia Study Group protocols between 1987 and 1993. Patients were classified into 5 groups according to chromosome number. The patients with a hyperdiploid(> 50) karyotype(13%) had the best prognosis [4-year event-free survival (EFS): 83 +/- 6%], while those with a pseudodiploid karyotype (24%) had the worst prognosis(4-year EFS: 52 +/- 6%) (log-rank, p = 0.03). However, multivariate analysis revealed that the ploidy classification had no prognostic significance in terms of EFS. When patients were classified according to chromosome abnormalities, those with any type of translocation had a worse outcome (4-year EFS: 33 +/- 9%) than those with hyperdiploidy(> 50), normal diploidy, and other abnormalities(log-rank, p < 0.0001). Multivariate analysis revealed that chromosome abnormalities were an independent prognostic factor (relative risk 3.98; p < 0.0001). Patients with t(1; 19) had an EFS similar to that of patients with chromosome abnormalities other than translocations or normal diploidy. We conclude that chromosomal findings have prognostic significance, although some chromosome abnormalities lost their statistical significance after modern intensified chemotherapy. Childhood ALL should be further stratified according to chromosome classification.

We report herein the clinical results of a multicenter trial of the Japan Adult Leukemia Study Group for cases of newly diagnosed acute promyelocytic leukemia (APL) treated with all-trans retinoic acid and chemotherapy (JALSG AML-92 study). Of 196 evaluable patients, 173 (88%) achieved complete remission (CR). Multivariate analysis showed that no or minor purpura at diagnosis and age less than 30 years were favorable factors for achievement of CR. There was a significant difference in the 4-year event-free survival between the AML-92 study (54%) and both the AML-87 (32%) and AML-89 (32%) studies which consisted of intensive chemotherapy. Since prognosis in patients with APL largely depends on chemotherapy, it is important to consider more effective chemotherapy during induction and consolidation therapy.

Mutation in the p53 tumor suppressor gene is known to play a critical role in the carcinogenesis of many types of human cancers, especially in colon carcinogenesis. To investigate the early stage of neoplastic processes, a simple and reliable method is needed to evaluate the state of p53 mutation. For this purpose, we tested the availability of the recently developed yeast functional assay to minute endoscopic samples. The assay consisted of 3 steps: 1) extraction of mRNA from the minute samples, 2) reverse transcriptional polymerase chain reaction (RT-PCR) amplification of the mRNA, and 3) transformation of yeasts by means of crude PCR products. This was an effective way to analyze many samples within a short period of time (2 days). The results were visible either as white colonies (wild type) or red colonies (mutant type). At first, we tested whether the quantity of mutants was measurable or not by analyzing a calibrated mixture of the cells known to harbor mutant and wild-type. The percentage of the red colonies that showed mutant p53 alleles was in proportion to the initial input percentage of the mutant p53 cells. Secondly, in clinical cases, it was found that minute samples (10-20 mg) were sufficient both for the analysis of yeast functional assays and pathologic examinations. The samples taken from normal mucosa and adenomas revealed fewer red colonies than the background value of this assay (10%). All the samples from adenocarcinomas yielded red colonies over 20%. The percentage of red colonies was well consistent with the ratio of cancer cell numbers to total cells. However, the percentage was not always correlated with the number of immunohistochemically positive cells stained with monoclonal antibody to detect abnormal p53. These results indicate that this assay combined with histological examination may provide useful information on determining carcinogenesis in endoscopic samples.

We examined 20 cases (21 specimens) of ocular adnexal lymphoid proliferations, using the histological, immunohistochemical and molecular genetic methods. The latter two protocols were performed to detect the light chains restriction of immunoglobulin with peroxidase-antiperoxidase (PAP) methods, and the clonality of immunoglobulin heavy chain gene with the hemi-nested polymerase chain reaction (PCR) method, respectively. Although in 8 cases it could not be morphologically determined whether they were neoplastic or not, clonality was revealed in 1 case with immunohistochemistry and in 4 cases with PCR. Two cases showed discordant results between immunohistochemistry and PCR probably due to somatic mutation of the framework region of the immunoglobulin heavy chain gene. Therefore, we, concluded that examination with these methods contribute to a better understanding of the nature of the ocular adnexal lymphoid proliferations. Furthermore, the immunoglobulin gene PCR method is very useful in practical examination, as it can be used with formalin-fixed paraffin-embedded specimens.

In yeast functional assay (YF assay), a newly developed screening system for p53 mutation, wild-type p53 gives white yeast colonies and transcriptionally inactive mutant p53 gives red colonies. In the present study, the author applied YF assay to the detection of p53 mutations in human oral squamous cell carcinoma (SCC). Total RNA was extracted from samples and YF assay was performed. Four SCC cell lines (SAS, HSC-2, HSC-3 and Ca9-22) known to have p53 mutations all gave 100% red colonies, whereas nine oral non-tumor tissues gave 2.9-10% (average 5.2 +/- 2.7%) red colonies. Furthermore, a rat hepatoma cell line, WHp53, which had been transfected with human wild-type p53 expression vector, presented 7.8% red colonies. Thus the functional assays of tissues or cells containing only wild-type p53 give 3-10% red colonies as a background. To assess the detectability of p53 mutations, YF assay was performed on mixtures of wild-type and mutant p53 PCR products at serial ratios. The result showed that the mutation was detectable if 6% population of transcriptionally inactive mutant p53 mRNA were present in the total p53 mRNA. Twenty-two clinical samples of human oral SCC were then tested by YF assay. Fourteen out of 22 cases gave more than 20% red colonies. In these 14 cases, clonal p53 mutations with deletion, nonsense mutation or missense mutation were identified. In a case which gave 17% red colonies, identical p53 mutation was found in 2 out of 6 independent red colonies. However, no identical mutations were found in the cases giving 13, 9 and 8% red colonies. Based on these results, the author proposes that 20% of red colonies is the minimal value for the diagnosis of p53 mutation in YF assay under PCR conditions using Pfu polymerase and hot start method.

We have developed a highly sensitive method to detect pelvic lymph node(LN) metastasis using reverse transcriptase-polymerase chain reaction(RT-PCR) with the primers specific for prostate-specific antigen(PSA) gene in combination with the fine needle aspiration biopsy(FNAB). The specimens were obtained from pelvic LN from 15 prostate cancer patients and 15 bladder cancer patients. The aspirated samples (0.05 approximately 0.1 ml) were used for detecting the fragment of PSA mRNA by RT-PCR and Southern blot analysis, and the rest of samples were submitted to conventional cytology. Expression of PSA gene was detected in 9 cases of FNAB samples including all 5 cytologically positive and further more 2 cytologically class III cases, and 2 of 8 cytologically negative cases. RT-PCR of FNAB samples from all cases of bladder cancer were negative for the detection of PSA gene. The sensitivity of PSA gene by RT-PCR was very high and could detect 10 degrees cancer cell. In conclusion, our study suggested that RT-PCR for detection of PSA gene in FNAB samples might become a new diagnostic tool for detection of small foci of prostatic cancer metastasis in LN and combination use of RT-PCR and cytology could greatly contribute to accuracy in diagnosis.

All-trans retinoic acid (ATRA) is widely used as a differentiation-inducing agent for acute promyelocytic leukemia (APL). However, one of the problems with this therapy is the difficulty in evaluating the extent of differentiation of leukemic cells. To determine objective indices for differentiation which can be used for judging complete remission, we treated 18 patients with acute promyelocytic leukemia, and performed sequential analysis of bone marrow, karyotype, RAR alpha gene rearrangement and PML-RAR alpha fusion gene transcript. The karyotype, RAR alpha rearrangement and PML-RAR alpha transcript returned to a normal pattern on days 31, 2, 34.8 and 47.6, respectively. We defined and analyzed maturation index (MI) by bone marrow findings as follows; (metamylocyte + band neutrophil + segmented neutrophil) (%)/(myeloblast + promyelocyte + myelocyte) (%). MI increased along with the differentiation of leukemic cells, and the days for MI exceeding 2 well correlated with the normalization of karyotype. These results suggested that MI can be used as an index of differentiation during ATRA therapy.

Fluorescence in situ hybridization was done with specimens obtained by bronchial brushing from 25 patients with abnormal lung shadows. A satellite DNA probe, specific for chromosome 11, was used to detect numerical chromosome aberrations in tumor cell nuclei. Normal diploid human lymphocyte nuclei, which served as control, had two signal spots in 99.6% of the nuclei in response to the chromosome 11 probe. The most frequent signal spots in class V cells (Case 1-7) ranged from 3 to 5, followed by 6 to 8, regardless of histopathological findings of lung cancer. In class I cells (cases 8-11) the signal appearance was similar to that in class V cells. The disease in patients from whom class I cells were obtained was found to be malignant by other diagnostic procedures performed afterward. The abnormalities in cases 13-25 were diagnosed as non-malignant by brush cytology, and clinical course showed a little more than 3 spots. These data indicate that fluorescence in situ hybridization with specimens obtained by bronchial brushing can be useful for detecting numerical chromosome abnormalities and can aid in the rapid diagnosis of lung cancer.

Twenty one patients with Philadelphia chromosome positive CML were treated with natural interferon alpha. All patients were in the chronic phase, 5 were untreated and 16 had been previously treated with busulfan or hydroxyurea. Eight patients in complete remission (CR) were given IFN subcutaneously at a dose of 5 x 10(6) unit per day as maintenance therapy, whereas 13 non-CR patients were given 2. 5 approximately 10 x 10(6) units for remission induction. Doses and intervals of IFN were adjusted to maintain the WBC count below 5 x 10(9)/l, but additional drugs were given when the WBC count could not be controlled with IFN alone. Six out of 10 evaluable non-CR patients attained CR with IFN only and 4 others achieved with additional drug. Cytogenetic responses were evaluated in 15 patients. CCR, PCR and MCR were attained in 5, 2 and 1 patients respectively. Southern blotting method showed that the BCR gene rearrangement disappeared in 5 out of 13 patients. Cytogenetic response rate was not different between untreated and previously treated patients, however it differed between patients with or without additional drug. The time to first cytogenetic effect was within 12 months in almost all effective cases. Fever and general fatigue were seen in almost all patients. IFN administration was discontinued only patients with severe skin eruption (3 patients) and bone marrow aplasia (1 patient).

The value of tumor angiogenesis, EGFR and c-erbB-2 oncoprotein, a long with p 53 protein expression for predicting relapse-free survival was investigated in 110 node-negative breast cancer patients. The grade of neovascularization was assessed by the microvessel density which was obtained by an immunocytochemical staining by factor VIII-related antigen. EGFR, c-erbB-2 oncoprotein and p 53 oncoprotein were also determined by immunocytochemical assay. Univariate analysis showed no statistical significance of EGFR, c-erbB-2 and p53 status as a prognostic indicator. However, the microvessel density was a significant predictor of relapse-free survival. Patients with over 100 counts of factor VIII-RA positive cells per mm2 field in the most active areas of neovascularization showed significantly poorer prognosis compared to those with less than 100 counts (p < 0.005). Multivariate analysis demonstrated that microvessel density was an independent prognostic indicator in node-negative breast cancer patients (p < 0.0005). It was suggested that microvessel density might be of use in selecting the high-risk group in node-negative breast cancer patients needing adjuvant therapies.

We analyzed the sensitizing activity of five agents, which have been assumed to be MDR-overcoming drugs, in two human renal cell carcinoma cell lines expressing the MDR1 gene at high levels. In addition, we studied the sensitizing activity of cepharanthin, an MDR-overcoming agents, to vinblastine, adriamycin, epirubicin, cisplatin, mitomycin C and etoposide. Based on the results, we treated 6 patients with metastatic renal cell carcinomas (4: bones, 2: contralateral kidneys) by intraarterial injection of vinblastine and adriamycin (or epirubicin) in combination with cepharanthin. Two of the 4 bone metastasis cases responded markedly to the treatment. Renal tubular impairment was observed in one patient who was treated for the contralateral kidney metastasis.

We treated 70 patients with acute promyelocytic leukemia (APL) with daily oral 45 mg/m2 all-trans retinoic acid (ATRA) in 2 multi-institutional prospective studies. Of 63 evaluable patients, 21 were resistant to initial induction chemotherapy, 10 were resistant to salvage chemotherapy after relapse, 17 were in the first relapse, 4 in the second relapse, 4 in the third relapse, and 7 were previously untreated. In the first study with ATRA from China, 18 (82%) of 22 evaluable patients achieved CR within 8 to 53 days with a median of 29 days. Initial peripheral leukemia cell counts were significantly less in the CR cases (p < 0.01). They were less than 100/cmm in 17 of 18 CR cases, and more than 200/cmm in all failure cases. Patients achieving CR received standard consolidation and maintenance chemotherapies, and the 16-month predicted continuing CR rate is 60%. Based on the first study, in the second study with ATRA from Hoffmann-La Roche AG, if initial peripheral leukemia cell counts were more than 200/cmm, chemotherapy was first given and then ATRA was started. Of 41 evaluable patients, 36 (88%) achieved CR within 11 to 91 days with a median of 34 days. Of 3 patients who received preceding chemotherapy due to high leukemia cell counts, 2 achieved CR. Morphological evidence of differentiation was noted in all CR cases, with Auer rods in mature segmented neutrophils in 13 cases. The clinical signs of DIC decreased rapidly within a few days and disappeared in CR cases. Toxicities attributable to ATRA were minimal and included cheilitis, xerosis, dermatitis, gastrointestinal disorders, bone pain, liver damage and high serum triglyceridemia.

Clinical trials of natural interferon-alpha (HLBI) in the treatment of Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) were carried out in a cooperative study of 22 institutions in Japan. Patients with CML in chronic and accelerated phase were given intramuscular or subcutaneous injections of HLBI at the dose of 6 X 10(6) U/body on consecutive days. Of the 47 patients in this study, forty-one were evaluable for clinical effects, and 42 could be assessed for adverse effects. Among 30 evaluable patients in the chronic phase, 9 (30.0%) achieved complete remission (CR) and 20 (66.7%) partial remission (PR). Only one patient had no response (NR), and the response rate (CR + PR) in chronic phase was 96.7% (29/30). Among 11 patients in the accelerated phase, 3 (27.3%) achieved CR and 4 (36.4%) PR. The response rate in the accelerated phase was 63.6% (7/11). In all 41 evaluable patients, the response rate was 87.8% (36/41). In 5 of 13 responding patients treated for more than 6 months, cytogenetic investigation showed the decline of Ph1 positive bone marrow cells from 100% to 92-0% (mean 46%). Adverse effects such as fever (52.4%), general fatigue (35.7%), and liver dysfunction (21.4%), were observed, but they were usually mild and reversible. No patient was taken off this study because of these toxicities. The results confirm the clinical efficacy of HLBI in patients with CML in the chronic and accelerated phase.