Yoichiro Yamamoto

Clinically relevant radioresistant cells efficiently repair DNA double‐strand breaks induced by X‐rays

Radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in radiotherapy and radiochemotherapy. Standard radiotherapy for tumor treatment consists of approximately 2 Gy once a day, 5 days a week, over a period of 5–8 weeks. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we established a novel radioresistant cell line, HepG2‐8960‐R with clinical relevance from parental HepG2 cells by long‐term fractionated exposure to 2 Gy of X‐rays. HepG2‐8960‐R cells continued to proliferate with daily exposure to 2 Gy X‐rays for more than 30 days, while all parental HepG2 cells ceased. After exposure to fractionated 2 Gy X‐rays, induction frequencies of micronuclei and remaining foci of γ‐H2AX in HepG2‐8960‐R were less than those in HepG2. Flow cytometric analysis revealed that the proportion of cells in S‐ and G2/M‐phase of the cell cycle was higher in HepG2‐8960‐R than in HepG2. These suggest that the response of clinically relevant radioresistant (CRR) cells to fractionated radiation is not merely an accumulated response to each fractionated radiation. This is the first report on the establishment of a CRR cell line from an isogenic parental cell line. (Cancer Sci 2009; 100: 747–752)

Long Incubation Period for the Induction of Cancer by Thorotrast is Attributed to the Uneven Irradiation of Liver Cells at the Microscopic Level

Abstract Yamamoto, Y., Usuda, N., Takatsuji, T., Kuwahara, Y. and Fukumoto, M. Long Incubation Period for the Induction of Cancer by Thorotrast is Attributed to the Uneven Irradiation of Liver Cells at the Microscopic Level. Radiat. Res. 171, 494– 503 (2009). Irradiation from internally deposited radionuclides induces malignant tumors. Ingested long-lived radionuclides accumulate in specific organs that are irradiated throughout life. To elucidate why the development of malignant tumors requires long-term internal exposure, of the order of decades, despite the fact that irradiation is continuous over this period, we analyzed intrahepatic cholangiocarcinoma in Thorotrast patients (Th-ICC). Autoradiography showed that the density of α-particle tracks was 50 times more concentrated than would be expected if Thorotrast were evenly distributed throughout the liver. The age–incidence curves revealed that while the incidence of hepatobiliary cancer in Japan increased in proportion to the 7th power of age, that of Th-ICC increased to the 6th power. Internal radiation significantly increased the randomness of hepatocyte distribution but not the density. Three major factors are considered to be responsible for the long incubation time: the uneven distribution of radionuclides, the limited range of radiation, and the movement of tumor precursor cells. Target cells susceptible to malignant transformation may undergo one event and may then migrate outside of the range of α particles, thereby avoiding immediate induction of successive additional events that would lead to cell death or neoplastic changes.

Supratentorial extraventricular anaplastic ependymoma in an adult with repeated intratumoral hemorrhage

We report the case of a 61-year-old man with supratentorial extraventricular anaplastic ependymoma who presented with repeated intratumoral hemorrhage. The patient was admitted with headache. Computed tomography and magnetic resonance imaging showed an enhancing mass with intratumoral hemorrhage in the right temporal lobe. Gross total resection was performed. The tumor was well demarcated from the brain tissue, and showed no continuity with the ventricular system. Histopathological examination revealed the features of anaplastic ependymoma. Therefore, additional radiation therapy and adjuvant chemotherapy were administered. Ten months later, the tumor recurred with hemorrhage in the spinal canal. This case showed rapid malignant progression and repeated intratumoral hemorrhage within a short period of time, both of which are characteristics of anaplastic ependymomas. Close observation of the central nervous system and adjuvant radiotherapy are mandatory, even if the ependymoma presents with repeated intratumoral hemorrhage.

Histological type of Thorotrast-induced liver tumors associated with the translocation of deposited radionuclides

Exposure to internally deposited radionuclides is known to induce malignant tumors of various histological types. Thorotrast, a colloidal suspension of radioactive Thorium dioxide (232ThO2) that emits alpha‐particles, was used as a radiographic contrast during World War II. Thorotrast is known to induce liver tumors, particularly intrahepatic cholangiocarcinoma (ICC) and angiosarcoma (AS), decades after injection. Therefore, patients injected with Thorotrast comprise a suitable study group to understand biological effects of internal ionizing radiation injury. Autoradiography and X‐ray fluorescence spectrometry (XRF) were carried out on non‐tumorous liver sections from Thorotrast‐induced ICC (T‐ICC) and Thorotrast‐induced AS (T‐AS). Autoradiography revealed that the slope of the regression line of the number of alpha tracks for the amount of deposited Thorium (232Th) was higher in non‐tumorous parts of the liver with T‐ICC than those with T‐AS. XRF showed that the intensity ratio of Radium (Ra) to Thorium (Th) in non‐tumorous liver tissue with T‐ICC was significantly higher than that with T‐AS. Furthermore, the mean 228Ra/232Th radioactivity ratio at the time of death calculated was also significantly higher in T‐ICC cases than in T‐AS cases. These suggest that the metabolic behavior of radionuclides such as relocation and excretion, as well as the content of deposited radionuclides, is a major factor in determining the histological type of Thorotrast‐induced liver tumors. (Cancer Sci 2009)

Tumour and immune cell dynamics explain the PSA bounce after prostate cancer brachytherapy

Background:Interstitial brachytherapy for localised prostate cancer may be followed by transient increases in prostate-specific antigen (PSA) that resolve without therapy. Such PSA bounces may be associated with an improved outcome but often cause alarm in the patient and physician, and have defied explanation.Methods:We developed a mathematical model to capture the interactions between the tumour, radiation and anti-tumour immune response. The model was fitted to data from a large cohort of patients treated exclusively with interstitial brachytherapy. Immunohistological analysis for T-cell infiltration within the same tumours was also performed.Results:Our minimal model captures well the dynamics of the tumour after therapy, and suggests that a strong anti-tumour immune response coupled with the therapeutic effect of radiation on the tumour is responsible for the PSA bounce. Patients who experience a PSA bounce had a higher density of CD3 and CD8 cells within the tumour that likely contribute to the PSA bounce and the overall better outcomes observed.Conclusions:Our observations provide a novel and unifying explanation for the PSA bounce in patients with early prostate cancer and also have implications for the use of immune-based therapies in such patients to improve outcomes.

Quantitative diagnosis of breast tumors by morphometric classification of microenvironmental myoepithelial cells using a machine learning approach

Machine learning systems have recently received increased attention for their broad applications in several fields. In this study, we show for the first time that histological types of breast tumors can be classified using subtle morphological differences of microenvironmental myoepithelial cell nuclei without any direct information about neoplastic tumor cells. We quantitatively measured 11661 nuclei on the four histological types: normal cases, usual ductal hyperplasia and low/high grade ductal carcinoma in situ (DCIS). Using a machine learning system, we succeeded in classifying the four histological types with 90.9% accuracy. Electron microscopy observations suggested that the activity of typical myoepithelial cells in DCIS was lowered. Through these observations as well as meta-analytic database analyses, we developed a paracrine cross-talk-based biological mechanism of DCIS progressing to invasive cancer. Our observations support novel approaches in clinical computational diagnostics as well as in therapy development against progression.

A novel method for morphological pleomorphism and heterogeneity quantitative measurement: Named cell feature level co-occurrence matrix

Background: Recent developments in molecular pathology and genetic/epigenetic analysis of cancer tissue have resulted in a marked increase in objective and measurable data. In comparison, the traditional morphological analysis approach to pathology diagnosis, which can connect these molecular data and clinical diagnosis, is still mostly subjective. Even though the advent and popularization of digital pathology has provided a boost to computer-aided diagnosis, some important pathological concepts still remain largely non-quantitative and their associated data measurements depend on the pathologist′s sense and experience. Such features include pleomorphism and heterogeneity. Methods and Results: In this paper, we propose a method for the objective measurement of pleomorphism and heterogeneity, using the cell-level co-occurrence matrix. Our method is based on the widely used Gray-level co-occurrence matrix (GLCM), where relations between neighboring pixel intensity levels are captured into a co-occurrence matrix, followed by the application of analysis functions such as Haralick features. In the pathological tissue image, through image processing techniques, each nucleus can be measured and each nucleus has its own measureable features like nucleus size, roundness, contour length, intra-nucleus texture data (GLCM is one of the methods). In GLCM each nucleus in the tissue image corresponds to one pixel. In this approach the most important point is how to define the neighborhood of each nucleus. We define three types of neighborhoods of a nucleus, then create the co-occurrence matrix and apply Haralick feature functions. In each image pleomorphism and heterogeneity are then determined quantitatively. For our method, one pixel corresponds to one nucleus feature, and we therefore named our method Cell Feature Level Co-occurrence Matrix (CFLCM). We tested this method for several nucleus features. Conclusion: CFLCM is showed as a useful quantitative method for pleomorphism and heterogeneity on histopathological image analysis.

Quantitative analysis of nuclear shape in oral squamous cell carcinoma is useful for predicting the chemotherapeutic response

The number of people afflicted with oral carcinoma in Japan has increased in recent years. Although preoperative neoadjuvant therapy with cisplatin and 5-fluorouracil are performed, chemotherapeutic response varies widely among the patients. With the aim of establishing novel indices to predict the therapeutic response to chemotherapy, we investigated the relationship between morphological features of pre-treatment oral carcinoma nuclei and the chemotherapeutic response using quantifying morphology of cell nuclei in pathological specimen images. We measured 4 morphological features of the nucleus of oral squamous cell carcinoma cases classified by the response to chemotherapy: No Change (NC) group, Partial Response (PR) group and Complete Response (CR) group. Furthermore, we performed immunohistochemical staining for p53 and Ki67 and calculated their positive rates in cancer tissues. Compactness and symmetry of the nucleus were significantly higher and nuclear edge response was significantly lower in cancer cells with lower chemotherapeutic responses compared high chemotherapeutic responders. As for positive rates of p53 and Ki67, there were no significant differences between any of the response groups. Morphological features of cancer cell nuclei in pathological specimens are sensitive predictive factors for the chemotherapeutic response to oral squamous cell carcinoma.

The uneven irradiation of a target cell and its dynamic movement can mathematically explain incubation period for the induction of cancer by internally deposited radionuclides.

Irradiation from internally deposited radionuclides induces malignant tumors. Ingested radionuclides accumulate in specific organs, which are irradiated over a lifelong period. Our aim is to elucidate why the development of malignant tumors requires long-term internal exposure, on the order of decades, despite the fact that irradiation is continuous over this period. Three major factors are considered to be responsible for the long incubation time in carcinogenesis caused by internally deposited alpha-emitters: uneven distribution of radionuclides, limited range of irradiation, and dynamic movement of tumor precursor cells. We hypothesized that target cells susceptible to malignant transformation may undergo one event by alpha particles and may then migrate outside of the range of alpha particles, thereby avoiding immediate induction of successive additional events that would lead to cell death or neoplastic changes. Based on this hypothesis, we further proposed a mathematical model to predict the relationship between dose rate and incubation period of tumors induced by internally deposited alpha-emitters. The function was non-linear and included terms of both direct and indirect radiation effects. It well fitted both human Th-ICC cases and rat 239Pu-induced lung cancer, suggesting that indirect radiation effects are independent from dose rate. The significance of parameters of the model is discussed.

An infant case of diffuse cerebrospinal lesions and cardiomyopathy caused by a BOLA3 mutation

INTRODUCTION Mitochondrial dysfunction results in a wide range of organ disorders through diverse genetic abnormalities. We herein present the detailed clinical course of an infant admitted for extensive, rapidly progressing white matter lesions and hypertrophic cardiomyopathy due to a BOLA3 gene mutation. CASE A 6-month-old girl with no remarkable family or past medical history until 1 month prior presented with developmental regression and feeding impairment. Ultrasound cardiography and brain magnetic resonance imaging (MRI) respectively disclosed the presence of hypertrophic cardiomyopathy and symmetrical deep white matter lesions. She was transferred to our hospital at age 6 months. High lactate levels in her cerebrospinal fluid suggested mitochondrial dysfunction. Despite vitamin supplementation therapy followed by a ketogenic diet, the patient began exhibiting clusters of myoclonic seizures and respiratory failure. Brain and spinal cord MRI revealed rapid progression of the white matter lesions. She died at 10 months of age. Fibroblasts obtained pre-mortem displayed low mitochondrial respiratory chain complex I and II activity. A homozygous H96R (c. 287 A > G) mutation was identified in the BOLA3 gene. DISCUSSION No reported case of a homozygous BOLA3 gene mutation has survived past 1 year of life. BOLA3 appears to play a critical role in the electron transport system and production of iron-sulfur clusters that are related to lipid metabolism and enzyme biosynthesis.