Significance of additive heat effect in the therapeutic gain factor in combined hyperthermia and radiotherapy: murine tumor response and foot reaction.

The thermal enhancement ratio (TER) and therapeutic gain factor (TGF) were evaluated for combined hyperthermia and radiation treatments of a murine fibrosarcoma, FSa-II. The TER is the ratio of the radiation dose that induces a given reaction without hyperthermia to that with hyperthermia. The TGF is defined as the ratio of TER for tumor response to TER for normal tissue response. Tumors in the subcutaneous tissue of the right foot were irradiated with graded radiation doses when they reached an average diameter of 6 mm (110 mm3). Hyperthermia was given by immersing animal feet in a constant temperature water bath 10 min before or after irradiation. The tumor growth time to reach 500 mm3 was obtained for each tumor and the median tumor growth time was calculated for each treatment group. For the normal tissue study, the non-tumor bearing murine foot was treated, as was the tumor, and the foot reaction was scored after treatment, according to our numerical score system for radiation damage, until the 35th post-treatment day and averaged. Using the fraction of animals showing a given average foot reaction score in a treatment group, the RD50, or the radiation dose to induce the given foot reaction or greater, was calculated. A single heating at 45.5 degrees C for 10 min and a step-down heating (first heat at 45.5 degrees C for 10 min immediately followed by the second heat at 41.5 degrees C for 60 min) prolonged the tumor growth time, indicating that hyperthermia per se resulted in some cell killing. The prolongation was greater following step-down heating than following single heating. These heat treatments alone induced no noticeable heat damage on the foot, but decreased the threshold dose observed on the radiation dose response curves for the foot reaction. Accordingly, TER and TGF were evaluated with or without normalizing this thermal effect. TER's for both tumor and foot responses without normalization were greater than the TER's after normalization and decreased with increasing radiation dose (between 1.9 and 7.1 or greater for tumor and between 1.3 and 4.3 or greater for foot reaction), whereas the normalized TER's were relatively constant (between 1.6 and 1.7 for tumor and between 0.7 and 1.5 for foot reaction). TGF's without normalization were greater than those obtained after normalization. The former was large at small doses and decreased with increasing radiation dose (between 1.5 and 4.0 or greater), whereas the latter was within 0.8 and 1.3 and relatively independent of radiation dose.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiation and thermal sensitivities of murine tumor (FSa-II) cells recurrent after a heavy irradiation.

Radiation and thermal sensitivities, and cell doubling times (Tds) of C3Hf/Sed mouse FSa-II cells recurring after a heavy irradiation were examined in vitro. Tumors in the leg were irradiated with gamma-rays and observed for late recurrence (in vivo clones), or removed immediately after irradiation and single cell suspensions were plated for colony formation (in vitro clones). Five subclones were selected from original cells in vitro. Survival curves were fitted to the multi-target and linear quadratic models. Surviving fractions at 2 (SF2) and 10 Gy (SF10) irradiations, and those at 30 and 60 min heatings at 44 degrees C (SF30 and SF60), were obtained for each clone. Although, Tds of subclones were slightly longer than those of the parental cells, those of recurrent clones were prolonged substantially with an exception of one cell line. Radiosensitivities of FSa-II parental cells tested in vitro and in vivo were equally radioresistant. Thermal sensitivities of parental cells tested in vitro and in vivo were also identical. All subclones were more radiosensitive compared to the parental cells. The in vitro recurrent clones showed smaller D0 (radiation dose to reduce survival from S to S/e in the exponential portion of survival curve) than the D0 of the parental cells. The SF2 values of four in vitro recurrent clones were greater than that of the parental cells whereas those of two lines were smaller. It was of interest that the in vivo recurrent tumor cells showed a wide variation in the radiation sensitivity. Among 9 tumor cell lines examined, 4 lines were more sensitive and 4 were more resistant compared to the original. FSa-II subclones as well as both in vitro and in vivo recurrent clones showed a wide variation in thermal sensitivity. No consistent changes in the shoulder or in the slope were found. The SF30 or SF60 showed that 5 out of 9 in vivo recurrent clones and 4 out of 9 in vitro clones were more resistant compared to the original cells. No correlation was observed between thermal and radiation sensitivities. The Td was not related with radiation or thermal sensitivity.

Enhancement of radiation effects by acyclovir.

Acyclovir (ACV), a new antiviral drug, was used to investigate its effect of radiosensitivity in tumors in vivo. In in vivo experiments with Sarcoma-180 transplanted into the ICR mouse and FM3A transplanted into the C3H mouse, ACV enhanced the radiosensitivity of both tumors. In S-180, radiation effects were enhanced by treatment with 100 mg/kg of ACV from 30 min before to 60 min after irradiation. In S-180 treated by 400 mg/kg of ACV, the enhancement ratio was approximately 2.0, as evaluated by the growth delay method. In the FM3A tumor treated by 20 mg/kg of ACV, the enhancement ratio was approximately 1.3, as evaluated by tumor cure (TCD50 assay). ACV is already clinically used as an antiviral drug. Its ability to radiosensitize tumors could therefore have clinical potential when combined with radiotherapy.

Mutation of the PTEN gene in advanced cervical cancer correlated with tumor progression and poor outcome after radiotherapy.

Improvement in management of advanced cervical cancer after radiotherapy requires a better understanding of its biological behavior. PTEN/MMAC/TEP(PTEN), a candidate tumor suppressor gene located at chromosome 10q23.3, was recently identified and found to be frequently mutated in several different types of human tumors. In contrast, rare mutations of the PTEN gene have been reported in cervical cancer. The aim of this study was to determine whether mutation of PTEN leads to increased genomic alteration in advanced cervical carcinoma, and to identify the correlation between mutation of PTEN and patient outcome after radiotherapy. We examined 50 primary advanced cervical carcinomas (37 patients of Stage IIIB, 13 patients of Stage IVA) treated with definitive radiotherapy using a PCR-based assay followed by SSCP and direct sequencing. The PTEN gene was mutated in 8 of the 50 (16%) patients (2 of Stage III, and 6 of Stage IV). There was a significant difference in Stage III versus IV between the wild-type PTEN patients and mutant PTEN patients (P=0.002). The tumor size was 6+/-2.1 cm in the wild-type PTEN tumors versus 8.5+/-2 cm in the mutant PTEN tumors (P=0.009). In addition, there was a significant difference in survival between the wild-type PTEN patients and mutant PTEN patients (P=0.009). The results of this study suggest that the PTEN gene mutation rate increases with tumor progression, and that the PTEN gene may play a role in both progression of cervical carcinoma and treatment outcome.

Multidetector-row CT analysis of time-dependent changes in lung fields after chest irradiation: usefulness of precision scans.

Time-dependent changes in lung fields after chest irradiation were analyzed using multidetector-row CT. Routine scans at 3-mm raw thickness and 8-mm recon thickness and precision scans at 0.5-mm raw thickness and 0.5-mm recon thickness were compared with respect to the number of each finding and the time-dependent changes in the rate of each finding. Among the findings visualized by these scans, ground-glass opacity (GGO) showed the highest overall appearance rate. Precision scans exceeded routine scans in the rates of all findings except GGO and confluent shadows, and the two types of scans showed the greatest difference in the rate of GGO. Since we found that GGO tended to be overestimated on routine scans, we confirmed it by a phantom experiment. Precision scans were similar or superior to routine scans in the rates of findings except 3 months after irradiation. We consider that the concomitant use of precision scans is useful in that it allows more accurate evaluation of various post-irradiation changes in lung fields including GGO, in which the lesion is in a reversible stage.

Polymorphism of the WAF1 gene is related to susceptibility to cervical cancer in Japanese women.

The WAF1 gene, located on chromosome 6p21.2, has been cloned and identified as a p53 mediator and an inhibitor of G1 cyclin-dependent kinases (CDKs). The present study was performed to investigate the possible role of the WAF1 gene in the pathogenesis of human cervical carcinoma. Matched venous blood and cancer tissues from 66 patients with cervical cancer were screened for WAF1 mutation by reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. A polymorphism in the WAF1 gene involving a cytosine (C) to an adenine (A) transversion at the third base of codon 31 was observed in 52 of 66 (78.8%) patients with cervical carcinoma and 68 of 108 (63%) normal individuals (P=0.02). A total of 7 patients (10.6%) were found to have a change of the WAF1 gene at codon 31 on comparison of blood and tumor specimens. An A-->C transversion in 5 tumors and a C-->A transversion in 2 tumors, that were not found in matching normal specimens, were observed. In addition, the presence of loss of heterozygosity (LOH) on chromosome 6p21.2 in tumor and normal tissue DNA were analyzed by PCR at three polymorphic microsatellite loci (D6S276, D6S1624, D6S1583). In two cases, there was a change of Arg/Ser in blood to Ser/Ser in the tumor, which did not involve LOH on 6p21.2. Therefore, somatic mutation of the WAF1 gene was detected in 3% (2 of 66) of patients with cervical cancer in this series. In conclusion, the increased frequency of WAF1 polymorphism in the patients studied implied that codon 31 Arg allele of the WAF1 gene may be associated with a tendency to develop cervical carcinoma. To our knowledge, this is the first report of WAF1 somatic mutations in primary human cervical cancer.

[Fundamental study on hyperthermic chemotherapy using adriamycin-loaded hydroxyapatite].

We developed a porous hydroxyapatite ceramic (HAP) incorporating adriamycin (ADM), that is, ADM-HAP as a new delivery system (DDS) to release ADM gradually. We also researched the possibility of hyperthermic chemotherapy using ADM-HAP by in vitro and in vivo experiments. As for in vitro experiments, we implanted HAPs into uniform Agar-Phantom, and observed thermal distribution generated by Thermotron-RF 8 using thermography. Then we found the hot spot that the edge temperature of HAPs always at the range of 0.5-0.7 degrees C than in the other regions. On the other hand, slow constant release (1%) of ADM from ADM-HAP in PBS was recognized for 24 hrs up to 30 days. When the incubating temperature was shifted up to 42.5 degrees C or 44 degrees C from 37 degrees C, the quantity released over 24 hrs increased about 1.1-fold or 1.3-1.4-fold of the cases at 37 degrees C, respectively. In the in vivo experiment, we inoculated Sarcoma 180 cells in the leg of ddY-mice, and measured the tumor growing times by the treatment of hyperthermia+ADM (whole body), hyperthermia+ADM (tumor region) or hyperthermia+ADM-HAP (tumor region). Then we found that the effect of hyperthermia with ADM-HAP inhibited synergistically the tumor growth as compared with hyperthermia with ADM. Consequently, we succeeded in tumor growth inhibition by increasing the temperature and by limiting ADM release to only a target region using hyperthermia with ADM-HAP.

[The modification of radiation effect by nucleoside related compounds on a murine tumor given with local radiotherapy and on survival following whole body irradiation].

We have evaluated the effect of nucleoside related compounds on a murine tumor given with local radiotherapy and on the survival rate following whole body irradiation. C3H/He mouse transplanted FM3A tumor and ddY mice were used. The enhancement ratio (ER) for the tumor treated by radiation and nucleoside related compounds was 1.02-1.67. For the normal tissue, Ara-A and BVAU given with radiation had no effect on the survival rate. In the combination of 50-800 mg/kg of ACV and whole body irradiation, the 50% survival day after 7 Gy was shorter than that of radiation alone. When 25-400 mg/kg of 3'-dG was given with radiation, the 50% survival day was longer. When combined with radiation, LD50/30 for ACV and 3'-dG were 3.9 and 6.3 Gy, respectively. ACV showed radiosensitizing effect with Dose modifying factor (DMF) at 1.1, on the other hand, 3'-dG showed radioprotective effect with DMF at 1.47. Our results suggested that some nucleoside analogues might be useful as radiosensitizer.

[Problems of antineoplastic effects by PLDR (potential lethal damage repair) inhibitor--pharmacokinetics].

PLDR is one known cause of tumor cell radioresistance. Drugs like ara-A have been reported to inhibit PLDR, thus increasing antineoplastic effects. In this research, ara-A concentration was measured by high-pressure liquid chromatography (HPLC) to investigate its metabolism. Ara-A deaminases in vitro in about 30 minutes, but by using a deaminase inhibitor such as 2'-deoxycoformycin, a fixed level of ara-A can be maintained. Furthermore, the new derivative, ara-AMP, does not deaminase. It is hoped that antineoplastic effects can be effectively increased by maintaining the ara-A concentration through the combined use of deaminase inhibitors and through new derivatives.

Chromosome 6p21.2, 18q21.2 and human papilloma virus (HPV) DNA can predict prognosis of cervical cancer after radiotherapy.

Loss of heterozygosity (LOH) is one of the most important mechanisms for inactivation of tumor-suppressor genes. Studies of LOH in patients with cervical carcinoma have reported a high frequency of LOH on 3p21.3, 6p21.2, 17p13.1, and 18q21.2. Our study explored whether p53 status, human papilloma virus (HPV), and LOH on chromosome 3p21.3, 6p21.2, 17p13.1, and 18q21.2 are associated with treatment outcome in 65 patients with cervical cancer after radiotherapy. Tumors and normal DNA were analyzed by polymerase chain reaction (PCR) for genetic losses at 10 polymorphic microsatellite loci. The presence of HPV and its type were analyzed by PCR-based assay using the consensus primers for E6, E7, and L1 region. Mutations of the p53 gene were identified by a single-strand conformation polymorphism analysis. Chromosomes 3p21.3, 6p21.2, 17p13.1, and 18q21.2 were involved in the LOH in 23.1%, 41.5%%, 33.8%, and 23.1% of the tumors in our study, respectively. HPV-positive tumors were found in 73.8% of the patients and p53 mutation in 10.8%. The patients with LOH on chromosome 6p21.2 and 18q21.2 survived significantly shorter compared with those without LOH on chromosome 6p21.2 and 18q21.2 in both the overall survival (P = 0.006 and P = 0.007) and the disease-free survival (P = 0.005 and P = 0.008). The HPV-negative patients survived significantly shorter compared with the HPV-positive patients in both the overall survival (P = 0.01) and the disease-free survival (P = 0.04). According to multivariate analysis, HPV status (P = 0.0004, P = 0.01), LOH on 6p21.2 (P = 0.006, P = 0.02), and LOH on 18q21.2 (in both P = 0.01) is a significant predictor of both overall and disease-free survival time. The results of our study suggest that absence of HPV infection, LOH on 6p21.2, and LOH on 18q21.2 are the most important determinants of outcome of patients with cervical carcinoma after radiotherapy.

A new liquid embolic material for liver tumors.

PURPOSE: To evaluate the feasibility of a new liquid embolic material, Onyx, for treating liver tumors. MATERIAL AND METHODS: Onyx is a mixture of 6% (w/v) ethylene-vinyl-alcohol copolymer dissolved in anhydrous dimethyl sulfoxide (DMSO) with 28% (w/v) tantalum powder. In addition to 6% Onyx, we also tried 4%, 2% and 1% solutions, prepared by adjusting the amount of DMSO. We used 15 white rabbits with liver tumors created by percutaneous injection of VX2 tumor cells. In 4 groups with 3 rabbits in each, the liver arteries were embolized with 6%, 4%, 2% and 1% Onyx, respectively, and in 3 rabbits DMSO alone was injected. The injections were performed just proximal to the bifurcation of the proper hepatic artery, followed by celiac arteriography. Post mortem, the livers were examined by soft-tissue radiography, and liver-tissue section microscopy. RESULTS: The maximum number of arterial branching points passed by embolic material in either the right or left hepatic arteries was 11, 15 and 16, for 6%, 4% and 2% Onyx, respectively, but was non-measurable for 1% Onyx. Minimum diameters of arteries reached by 6%, 4%, 2% and 1% Onyx in tumorous areas were 40 microm, 35 microm, 20 microm and 10 microm, respectively, and in non-tumorous areas 35 microm, 5 microm, 5 microm and 5 microm, respectively. CONCLUSION: This study suggests that Onyx may be feasible for treatment of hepatic tumors.

Implantation treatment method of slow release anticancer doxorubicin containing hydroxyapatite (DOX-HAP) complex. A basic study of a new treatment for hepatic cancer.

We performed an experimental study on slow releasing anticancer drug implantation treatment as a new therapy for hepatocellular carcinoma. Hydroxyapatite (HAP) was chosen for the carrier material and doxorubicin hydrochloride (DOX) for anticancer agent. DOX-HAP was produced by adsorbing DOX to porous HAP particles of 1375 +/- 125 microns diameter using the freeze drying method. In vitro experiments showed slow release of the drug resulting in the steady release of DOX from HAP for 1 month duration. In healthy white rabbits with DOX-HAP implantation in the liver, serum DOX was not detectable, and DOX release rate was stable at the implanted region after 7, 14, and 21 days. When DOX-HAP (DOX; 100 mg kg-1) was administered to mice with sarcoma 180, an improved survival rate was observed without acute toxicity. We also found that VX2 liver tumour growth on white rabbit was inhibited by implantation of DOX-HAP, without acute toxicity. We hope that DOX-HAP implantation therapy will open up new avenues for the treatment of hepatoma.