Minoru Suzuki

Prospective trial of concurrent chemoradiotherapy with protracted infusion of 5-fluorouracil and cisplatin for T4 esophageal cancer with or without fistula.

PURPOSEnA prospective trial of concurrent chemoradiotherapy (CT-RT) with a protracted infusion of 5-fluorouracil and cisplatin was performed to evaluate the safety and efficacy of this protocol for T4 esophageal cancer (UICC 1997).nnnMETHODS AND MATERIALSnBetween 1998 and 2000, 28 patients with T4 esophageal squamous cell carcinomas were treated with concurrent CT-RT. Of the 28 patients, 15 had Stage III, 5 Stage IVA, and 8 Stage IV disease. Five of the T4 tumors had evidence of fistula before treatment. Patients received a protracted infusion of 5-fluorouracil 300 mg/m(2)/24 h on Days 1-14, a 1-h infusion of cisplatin 10 mg/body on Days 1-5 and 8-12, and concurrent radiation at a dose of 30 Gy in 15 fractions during 3 weeks. This schedule was repeated twice, with a 1-week split, for a total RT dose of 60 Gy during 7 weeks for 25 patients. For the remaining 3 patients, 30 Gy of preoperative CT-RT was administered.nnnRESULTSnOf the 25 patients who were treated with the full dose of CT-RT, 14 (56%) completed the two courses of the CT-RT protocol, and 8 patients (32%) received the full dose of RT but a reduced dose of chemotherapy. Eight (32%) of the 25 tumors showed complete regression. Although Grade 3 hematologic toxicities were frequently noted, Grade 4 or more hematologic toxicities were few. Of the 5 T4 fistulous tumors, 2 demonstrated the disappearance of the fistula after CT-RT. However, the worsening or development of an esophageal fistula was noted in 5 patients. The 2-year survival rate for patients with Stage III was 27%, and the median survival time for those with Stage III and Stage IVA+IV was 12 and 5 months, respectively.nnnCONCLUSIONnDespite its significant toxicity for esophageal fistula, this concurrent CT-RT protocol of protracted 5-fluorouracil infusion and cisplatin appears feasible and effective for T4 esophageal cancer with or without fistulas.

Radiosensitization effect by combination with paclitaxel in vivo, including the effect on intratumor quiescent cells.

PURPOSEnTo evaluate the radiosensitization effect on solid tumors upon combination treatment with paclitaxel (TXL), including the effect on intratumor quiescent (Q) cells.nnnMETHODS AND MATERIALSnMice bearing SCC VII or EL4 solid tumors received 5-bromo-2-deoxyuridine (BrdU) continuously for 5 days to label all proliferating (P) cells. The mice then received gamma-irradiation with or without tirapazamine (TPZ) at various time points after TXL administration. Another group of mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors at various time points after TXL administration. Immediately after irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, the tumor cells were isolated from the solid tumors in another group of mice, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN or apoptosis frequency of Q cells was then used to calculate the surviving fraction of Q cells from the regression line for the relationship between the MN or apoptosis frequency and the surviving fraction of total tumor cells.nnnRESULTSnIn both SCC VII and EL4 tumors, maximum values of mitotic index (MI) and apoptosis frequency were observed 9 and 24 h after TXL administration, respectively. However, on the whole, the apoptosis frequency for SCC VII was very low. gamma-Irradiation 9 h after TXL administration induced significant radiosensitization effects on the total cells of both tumors. Irradiation at 60 h had a more significant effect on total cells of EL4 tumor, but no significant effect on total cells of SCC VII tumor. Combined treatment with TXL induced no radiosensitization effect on Q cells in either tumor. The effect on Q cells was observed only after TPZ was administered. The HF of total cells in EL4 tumors decreased significantly 60 h after TXL administration.nnnCONCLUSIONnNo radiosensitization effect upon combination treatment with TXL is induced in Q tumor cells. However, the effect on P cells is produced by irradiation at the time when the maximum values of MI are induced following TXL administration. In addition, for tumors that are susceptible to apoptosis after TXL administration alone, irradiation at the time of sufficient reoxygenation in tumors after TXL administration produces a greater radioenhancement effect on P cells.

Evaluation of apoptosis and micronucleation induced by reactor neutron beams with two different cadmium ratios in total and quiescent cell populations within solid tumors.

PURPOSEnResponse of quiescent (Q) and total tumor cells in solid tumors to reactor neutron beam irradiation with two different cadmium (Cd) ratios was examined in terms of micronucleus (MN) frequency and apoptosis frequency, using four different tumor cell lines.nnnMETHODS AND MATERIALSnC57BL mice bearing EL4 tumors, C3H/He mice bearing SCC VII or FM3A tumors, and Balb/c mice bearing EMT6/KU tumors received 5-bromo-2-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty min after i.p. injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutron beams. The tumors without 10B-compound administration were irradiated with neutron beams or gamma-rays. This neutron beam irradiation was performed using neutrons with two different Cd ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the MN frequency in cells without BrdU labeling (=Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, for apoptosis assay, 6 h after irradiation, tumor cell suspensions obtained in the same manner were fixed, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequencies in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU.nnnRESULTSnWithout 10B-compounds, the sensitivity difference between total and Q cells was reduced by neutron beam irradiation. Under our particular neutron beam irradiation condition, relative biological effectiveness (RBE) of neutrons was larger in Q cells than in total cells, and the RBE values were larger for low Cd-ratio than high Cd-ratio neutrons. With 10B-compounds, both frequencies were increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH did. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of Q cells treated with BSH. Whether based on the MN frequency or the apoptosis frequency, similar results concerning the sensitivity difference between total and Q cells, the values of RBE, and the enhancement effect by the use of 10B-compound were obtained.nnnCONCLUSIONnApoptosis frequency, as well as the MN frequency, can be applied to our method for measuring the Q cell response to reactor neutron beam irradiation within solid tumor in which the ratio of apoptosis to total cell death is relatively high, as in EL4 tumor. The absolute radiation dose required to achieve the same endpoint for Q cells is much higher than that for total cells when combined with 10B-compound, especially with BPA.

Erythropoietin/Erythropoietin-receptor system as an angiogenic factor in chemically induced murine hepatic tumors

BackgroundTo clarify the role of erythropoietin (Epo) in hepatic tumor angiogenesis, expression of Epo and its receptor (Epo-R) and content of Epo were investigated in murine chemically induced hepatic tumors.MethodsTo induce hepatic tumors and cirrhosis, diaminobenzidine was administered to Wistar rats for 5 months. In total, 30 hepatic tumors of greater than 3u2009mm in diameter were induced in 12 rats. The 30 hepatic tumors were resected with the surrounding hepatic tissues. The Epo content was measured by a radioimmunoassay (RIA) method. The number of tumor vessels in a definite area was counted in 100 areas of each tumor. To demonstrate the expression of Epo-R in tumors or surrounding liver tissues, immunohistochemial staining for Epo-R was performed.ResultsThe Epo content of tumors ranged from 6.1 to 97.8u2009mU/ml, with a median of 21.8u2009mU/ml, which was significantly higher than that of the cirrhotic tissues adjacent to the tumors. Epo was not detectable in the normal or cirrhotic liver tissues without tumors. A significant correlation between Epo content and vascular density was noted in the 30 hepatic tumors (correlation coefficient, 0.480; P = 0.01). Immunoreactive Epo-R was detectable in the endothelium of intervening vessels of all hepatic tumors examined.ConclusionThe Epo/Epo-R system is related to the angiogenesis of murine hepatic tumors.

Radiobiologic significance of apoptosis and micronucleation in quiescent cells within solid tumors following γ-ray irradiation

PURPOSEnTo determine the frequency of apoptosis in quiescent (Q) cells within solid tumors following gamma-ray irradiation, using four different tumor cell lines. In addition, to assess the significance of detecting apoptosis in these cell lines.nnnMETHODS AND MATERIALSnC3H/He mice bearing SCC VII or FM3A tumors, Balb/c mice bearing EMT6/KU tumors, and C57BL mice bearing EL4 tumors received 5-bromo-2-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received gamma-ray irradiation at a dose of 4--25 Gy while alive or after tumor clamping. Immediately after irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 hours after irradiation, tumor cell suspensions obtained in the same manner were fixed. The apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU.nnnRESULTSnIn total cells, SCC VII, FM3A, and EMT6/KU cells showed reasonable relationships between MN frequency and surviving fraction (SF). However, fewer micronuclei were induced in EL4 cells than the other cell lines. In contrast, a comparatively close relationship between apoptosis frequency and SF was found in total cells of EL4 cell line. Less apoptosis was observed in the other cell lines. Quiescent tumor cells exhibited significantly lower values of MN and apoptosis frequency probably due to their large hypoxic fraction, similar to total tumor cells on clamped irradiation.nnnCONCLUSIONngamma-ray irradiation induced MN formation in SCC VII, FM3A, and EMT6/KU tumor cells, and the apoptosis was marked in EL4 cells compared with the other cell lines. Our method for detecting the Q cell response to gamma-ray irradiation using P cell labeling with BrdU and the MN frequency assay was also applicable to apoptosis detection assay.

COMBINED EFFECTS OF TIRAPAZAMINE AND MILD HYPERTHERMIA ON ANTI-ANGIOGENIC AGENT (TNP-470) TREATED TUMORS—REFERENCE TO THE EFFECT ON INTRATUMOR QUIESCENT CELLS

PURPOSEnTo evaluate the efficacy of the use of tirapazamine (TPZ), especially combined with mild hyperthermia (40 degrees C, 60 min), in the treatment of solid tumors following an anti-angiogenic treatment with TNP-470. In addition, we assessed the effect of TPZ and/or mild hyperthermia (MHT) combined with conventional radiotherapy or chemotherapy on TNP-470 treated tumors.nnnMATERIALS AND METHODSnC3H/He mice bearing SCC VII tumors subcutaneously received TNP-470 at two doses of 100 mg/kg after tumor cell inoculation. At the same time, the tumor-bearing mice received 5-bromo-2-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received TPZ administration combined with or without MHT, gamma-ray irradiation combined with or without TPZ and/or MHT, or cisplatin injection with or without TPZ and/or MHT. Another group of mice received a series of test doses of gamma-rays while alive or after being killed to obtain hypoxic fractions (HFs) in the tumors at various time points after the above-mentioned cytotoxic treatment point. After each treatment, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (or quiescent [Q] cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells.nnnRESULTSnTPZ administration combined with TNP-470 treatment and MHT increased the MN frequency more markedly than treatment with TPZ alone, and this tendency was more remarkable in Q cells than total cells. In both total and Q cells, combined treatment with TPZ and MHT produced significant increases in MN frequencies whether gamma-rays were delivered to TNP-470 treated tumors or cisplatin was injected into the TNP-470 administered mice. Although not significantly, the HFs of total and Q cell populations within solid tumors increased after TNP-470 treatment.nnnCONCLUSIONnCombined treatment with TPZ and MHT, whether other cytotoxic treatments such as gamma-ray irradiation or chemotherapy using cisplatin were combined or not, was useful for sensitizing tumor cells in vivo including Q cells even after TNP-470 treatment.

The Combined Effect of Electroporation and Borocaptate in Boron Neutron Capture Therapy for Murine Solid Tumors

10B‐Enriched borocaptate (BSH) was administered intraperitoneally to SCCVII tumor‐bearing C3H/He mice. Electroporation (EP) was conducted by using a tweezers‐type electrode. The 10B contents in tumors were measured by prompt γ‐ray spectrometry. The colony formation assay was applied to investigate the antitumor effects of boron neutron capture therapy (BNCT) and thereby to estimate the intratumor localization of BSH. The 10B concentrations in tumors decreased with time following BSH administration, falling to 5.4(±0.1) ppm at 3 h, whereas EP treatment (3 repetitions) 15 min after BSH injection delayed the clearance of BSH from tumors, and the 10B level remained at 19.4(±0.9) ppm at 3 h. The effect of BNCT increased with the 10B concentration in tumors, and the combination with EP showed a remarkably large cell killing effect even at 3 h after BSH injection. The effect of BNCT, i.e., slope coefficient of the cell survival curve of tumors, without EP was proportional to tumor 10B level (r=0.982), and that of BSH‐BNCT combined with EP lay close to the same correlation line. However, tumors subjected to EP after BSH injection did not show high radiosensitivity when irradiated after conversion to a single cell suspension by enzymatic digestion. This indicates that the increase of the BNCT effect by EP was a consequence of enclosure of BSH in the interstitial space of tumor tissue and not within tumor cells. This is different from a previous in vitro study. The combination of EP and BNCT may be clinically useful, if a procedure to limit EP to the tumor region becomes available or if an alternative similar method is employed.

Comparison of outcomes between overlapping structure-based and non-overlapping structure-based optimization for simultaneous integrated boost IMRT for malignant gliomas

BackgroundIntensity-modulated radiotherapy (IMRT) can deliver different doses to two target volumes with high conformity. The purpose of the present study was to compare outcomes provided by two different optimization methods, overlapping structure-based and non-overlapping structure-based methods, for simultaneous integrated boost (SIB)-IMRT for malignant gliomas.MethodsTreatment plans for three glioblastomas and one anaplastic astrocytoma were analyzed in the present study. The planning protocol was to deliver 70u2009Gy/28 fractions (fr) to the gross tumor volume (GTV) and 56u2009Gy/28u2009fr to the surrounding edema. Two different optimization methods were tested for optimizing dose distribution to the GTVand the surrounding edema. One method was the “including method”, an overlapping structure-based (GTV and the clinical target volume [CTV]) optimization method. The other method was the “annulus method”, a non-overlapping structure-based (GTV and the subtracted volume) optimization method. Dosimetric indexes derived from dose-volume histograms (DVHs) were used for the analysis.ResultsThere was no significant difference between the two methods in the mean doses of the target volumes and the doses delivered to the 5% or 95% target volumes (D05 or D95). The mean dose to the brain by the including method was significantly higher than that delivered by the annulus method (P = 0.0001). The D05 of the brain showed no significant difference between the two methods.ConclusionThe two optimization methods provided comparable dose distributions within the target volumes and normal brain.

Changes in the Sensitivity of Intratumor Cells during Fractionated Tirapazamine Administration

Mice bearing solid tumors received 10 intraperitoneal administrations of 5‐bromo‐2′‐deoxyuridine (BrdU) to label the proliferating (P) tumor cells. Then, as a priming treatment, tirapazamine (TPZ) was intraperitoneally administered. Further, 0 through 48 h later, the tumor‐bearing mice received TPZ again at various doses. The tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequencies in cells with and without BrdU labeling, which were regarded as P and quiescent (Q) cells at the priming treatment, respectively, were determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. In addition, P cell ratios in the tumors at the second treatment were determined using immunofluorescence staining for P cell nuclear antigen. In each cell fraction, the longer the interval between the two treatments, the higher was the sensitivity to TPZ, except 1 h after the priming treatment. More than 24 h later, total and P cells, especially P cells, showed significantly higher sensitivity to TPZ than in the case of a single TPZ treatment. The longer the period between the two TPZ treatments, the lower was the P cell ratio at the second treatment. These findings were thought to indicate that the use of TPZ in the treatment of solid tumors causes a shift from the P to the Q state in vivo.

Boron Neutron Capture Therapy of Malignant Gliomas

Boron neutron capture therapy (BNCT) is a promising modality for biochemically targeted, highly selective radiation treatment of various cancers, including malignant gliomas. Currently available results demonstrate the beneficial effect of such therapy on survival of patients with both recurrent and newly diagnosed glioblastomas. The main drawback of BNCT in cases of previously irradiated neoplasms is high rates of symptomatic pseudoprogression and radiation necrosis. For prevention of these complications, concurrent administration of bevacizumab may be helpful. Further studies are needed to establish the optimal therapeutic protocols and to define the exact role of this management option in multimodality treatment strategies. Recent technological developments of accelerator-based neutron sources may simplify placement of the device for BNCT within clinical facilities and lead to wider application of this technique in cases of various cancers.