Corynebacterium parvum-induced radiosensitivity and cycling changes of hematopoietic spleen colony-forming units.

Ten days after total-body irradiation with 550 rads of b0Co, spleen colonies were observed in adult C57BL mice. A change in radiosensitivity induced by Corynebacterium parvum, as measured by increased numbers of colony-forming units that survived the 550 rads, began shortly after C. parvum stimulation and extended for at least 7 days before irradiation. C. parvum given 4-24 hours before, followed by high specific activity [3H]thymidine (HSATT) 1 hour before total-body irradiation greatly reduced survival of the stem cells that formed spleen colonies (CFUs) and CFUs radiosensitivity to control levels. The HSATT sensitivity by "suicide" assay in vivo and the time-response change in radiosensitivity corresponded with the decrease in radiosensitivity, which showed that CFUs were stimulated by C. parvum administration and entered the S-phase shortly after stimulation. The data indicated a resting population close to the S-phase. After stimulation, this population entered S-phase. Syngeneic mouse lymphoma cells injected iv 24 hours earlier did not elicit any effect as a stimulus to CFUs radiosensitivity change.

Combined modality treatment using radiation and/or chemotherapy in an athymic nude mouse-human medulloblastoma and glioblastoma xenograft model.

A human medulloblastoma (BN-2) and a glioblastoma (BN-3) which were previously established in nude mice were used to determine the effect of combined modality therapy with gamma-radiation, and three chemotherapeutic agents, procarbazine, 1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo diethylester (AZQ), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The tumor cells were grown in tissue culture and implanted intracranially in the right cerebral hemisphere of NIH Swiss nude mice to a depth of 3 mm. The mice were randomized, and treatment was started 3 days after tumor implantation. Procarbazine and AZQ were injected i.p. every 5 days for three treatments. BCNU was injected one time for a single treatment. Radiation was localized to the head. A 60Co unit was used for irradiation at the rate of 125 rads/min 3 days after tumor implantation. Ten experiments were performed using six to nine mice per group and different drug-radiation dose combinations. The drug dose ranged from 400 to 500 mg/kg/injection for procarbazine, 7.5 mg/kg/injection for AZQ, and 10 to 20 mg/kg/injection for BCNU. The radiation dose ranged from 320 to 1050 rads/mouse (whole head). The day of death was recorded for each animal, and the mean of each treatment group was used to calculate the percentage increase in life span (ILS) compared to the untreated control group. Chemotherapy alone produced a minimal effect, while radiation alone produced minimal effects at 320 to 640 rads with progressively positive effects at 800 and 1050 rads. When the combination treatment of the human medulloblastoma xenograft with procarbazine was used, the ILS was significantly increased in all four experiments, ranging from 25 to 41%, and was superior to single-modality treatment in all but the 1050-rad treatment, where it showed an equal effect. The combination treatment using AZQ and BCNU showed no ILS for the medulloblastoma tumor. Combination treatment of the human glioblastoma xenograft using BCNU produced significant ILSs of 105 and 119% and was superior to single-modality treatment with a drug dose of 10 mg/kg and radiation doses of 540 and 800 rads, respectively. The nude mouse-human tumor xenograft model was found to be useful for combined modality studies and should give valuable information for the experimental design of pilot Phase III clinical studies against a variety of brain tumors.

Regeneration in cervix cancer after 252Cf neutron brachytherapy.

Regeneration of clonogens in human cervical cancer was assessed by the pathological evaluation of the hysterectomy specimen after intracavitary 252Cf neutron brachytherapy implants separated by varying time intervals followed by extrafascial hysterectomy. In this study, patients with bulky/barrel shaped Stage IB cervical cancers received 252Cf implants plus approximately 45 Gy of whole pelvis linear accelerator radiotherapy in approximately 25 fractions in 5 weeks followed by hysterectomy 4-6 weeks after radiotherapy. The specimens were studied grossly and microscopically for residual tumor. It was found that the fraction of positive specimens increased with elapsed time interval between implants. These findings support the hypothesis that there is repopulation of surviving clonogens with increased time interval between the implants. The observation also supports current concerns that rapid depopulation of tumor can lead to rapid repopulation, that is, rapid shrinkage of tumor can alter the physiological environment such that clonogens can rapidly regenerate.

A tumor/normal tissue advantage for low dose rate neutron brachytherapy.

Reports on clinical study of Cf-252 pelvic brachytherapy are reviewed and show that complication frequency is low. Low dose rate (LDR) neutron brachytherapy has been shown to be effective against cervical and advanced pelvic cancers; and produces 5 year cures without a high frequency of normal tissue complications. This is attributed to a high relative biological effectiveness (RBE) for the bulky, hypoxic tumor which along with an oxygen enhancement ratio (OER) advantage and dose-rate independent effects, produces rapid tumor regression and good local tumor control. Adjacent normal tissues which are oxygenated have lower RBE values than that of hypoxic tumors. Cf-252 brachytherapy increases the dose differential, therapeutic gain and the probability of local tumor control, since the high RBE of Cf-252 for hypoxic tumor is much less in normal tissues. Cf-252 radiation concentrated in the tumor, and for this reason, has had much fewer attendant normal tissue complications compared to neutron beam therapy.

Study of biological effects of varying mixtures of Cf-252 and gamma radiation on the acute radiation syndromes: relevance to clinical radiotherapy of radioresistant cancer.

PURPOSE: Data for the 30 day bone marrow syndrome (BM-50) and the 6-10 day gastrointestinal (GI-50) syndrome for a one and two fraction schedule and acute and low dose rate irradiation using pure and mixed Cf-252 and photon radiation are presented. METHODS AND MATERIALS: The radiations of Cf-252 is a mixture of neutrons and gamma rays. We total body irradiated Balb/c mice of both sexes with acute Co-60, low dose rate Cs-137 and Cf-252 using a 1 x and 2 x schedule. For low linear energy transfer radiations of Co-60 or Cs-137 there was expected to be an increase in the dose to produce the gastrointestinal and bone marrow syndromes with minimal change for Cf-252 neutrons. However, the radiations from Cf-252 are approximately 65% neutrons and approximately 35% photons and hence some repair may be expected. We further altered the proportion of photons in the Cf-252 radiation field by mixing Cs-137 with the Cf-252 sources and total body irradiated the mice to determine the effects on the syndromes. We determined the effects of mixing Cf-252 neutrons with different proportions of photons on the radiation syndromes. RESULTS: There was increase in BM-50 and GI-50 doses with fractionated or low dose rate photon irradiations and the dose modifying factors were 1.3-1.4 for the GI syndrome and 1.2 for the bone marrow syndrome. For Cf-252 there was minimal fractionation effect for the GI-50 syndrome, which increased by a 1.1 for x 1 vs. x 2 fractions; for the BM-50 syndrome it rose by a 1.1 factor. For LDR Cs-137 the dose for the GI-50 syndrome rose by a 2.2 fold. For mixed neutron-photon radiation of 0%, 15%, 35%, and 65% eta/gamma mixtures, the dose to produce the BM-50 and GI-50 endpoints dropped sharply from 0 to 35% neutrons and remained flat thereafter. CONCLUSION: For major tissues such as the bone marrow and G-I tract, Cf-252 behaved as high linear energy transfer for mixtures of neutrons and gamma rays of approximately 35% neutrons when the radiation were delivered simultaneously at the low dose rates studied. There was little or no additional contribution to the effectiveness of the mixed radiations if neutrons contributed 35% or more of the dose.

Cf-252 leukemogenesis in the C57BL mouse.

Radiation-induced leukemia/lymphomas were induced in C57BL mice using four weekly acute 60Co fractionated irradiation exposures (to 188 cGy, or graded doses of low dose rate (LDR) Cf-252 irradiation given in fractionated exposure sessions at four weekly intervals. The acute 60Co radiation produced 84% thymic lymphomas with a median survival time (MST) of 162 days for mice developing tumors. Mice were exposed to Cf-252 n + gamma radiation in graded doses of 50, 62.5, 80, 112, and 188 cGy per week repeated 4X. Mice exposed to Cf-252 radiation developed thymic lymphomas on a much delayed time schedule. Mice irradiated at 50-80 cGy Cf-252 were killed after the 60Co induced thymoma mice had died to detect tumors. At Cf-252 doses of 112 or 188 rads 79 or 70% of mice, respectively, developed thymic lymphomas and had similar survival times which gave an estimated leukemogenesis RBEn of approximately 1.0-2.0. These studies show that for Cf-252 n + gamma radiation, compared to 60Co for leukemogenic efficiency, had a much longer latent period, and had a low RBE (1.0-2.0) at the large doses per fraction used in these studies. Under the experimental fractionated conditions tested, Cf-252 neutrons were leukemogenic, but only slightly more so than fractionated 60Co.

RBE of CF-252 neutrons by mouse testes weight loss.

Mouse testes weight decreased with increasing radiation dose following exposure to Cf-252 and its mixed neutron plus gamma radiation. The dose response for low dose rate (LDR) Cf-252 followed two components on an exponential plot of log percent weight loss vs. dose. Similar dose-response relationships were observed for acute 60Co or LDR Cs-137 radiations, but these required much larger doses to produce the same effect. The RBE was calculated against acute 60Co and Cs-137. There were no differences between acute 60Co and LDR Cs-137 effects. Cf-252 neutron RBE (n + gamma) was 3.7 for testes weight loss. RBEn for the neutrons-only was 5.1 RBE for the radiosensitive portion was 4.0.

RBE of spleen CFU-S to low dose rate Cf-252 or photon radiation in vivo.

The biological effects of low dose rate (LDR) Cf-252 radiation were compared to LDR Cs-137 photon radiation and acute 60Co radiation. The RBEn for endogenous CFU-S in vivo was 2.1 for neutron radiation at a dose rate of 9.9 cGy/hr of Cf-252 radiation. The value was consistent with previous in vitro experiments where a value of 2.1 was found. For the low doses and low dose rates studied, we tested an acute assay dose following the LDR irradiation to determine dose-effect and RBEn. Organ size shrinkage and regeneration patterns after LDR Cf-252 and Cs-137 were also studied and showed greater growth delay for Cf-252 irradiated lympho-hemopoietic tissues.

Study of acute 60Co, low dose rate CF-252 and CS-137 radiation on LSA ascites lymphoma in vivo.

Dose response curves were determined for the LSA lymphoma for acute 60Co, low dose rate Cs-137 and Cf-252 radiations using in vivo survival time bioassay. Mean survival times increased with dose with a prominent oxygen effect noted for acute 60Co and Cs-137. OER was lowest for Cf-252 where it was approximately 1.4. The RBEn for oxic LSA cells to Cf-252 neutrons was 3.1 for acute 60Co and 4.2 for Cs-137. It was larger for hypoxic tumor and RBE was 5.3 for 60Co and 5.8 for Cs-137. Survival curves based on survival data used a multitarget dose-response model for photon radiation and exponential dose-response for Cf-252 radiation. When LSA was irradiated in advanced tumor stages in vivo, Cf-252 was much more effective than acute 60Co or LDR Cs-137 for increasing survival time. Tumor response in vivo matched the in vitro irradiated tumor data. No schedule dependence was observed for mixing of 60Co and Cf-252 radiations.

Clinical trial of 252Cf neutron brachytherapy vs. conventional radiotherapy for advanced cervical cancer.

252Cf, a neutron emitting radioactive transplutonium isotope, was tested for its efficacy against advanced bulky Stage III-IV cancers of the cervix in a clinical trial at the University of Kentucky Medical Center. Eighty-two patients were treated during 1976-1979 and followed for 5-year survival and tumor control. Three different treatment methods went on sequentially and concurrently, that is, (a) conventional whole pelvis photon with delayed 137Cs implants, (b) conventional photon therapy with delayed 252Cf implants, and (c) 252Cf implants ("early") preceding photon therapy. There were 12% 5-year survival for Stage IIIB cancers by conventional therapy, and 15% by delayed 252Cf implant therapy. For early 252Cf implant therapy there were 54% 5-year survivals with 4% complications and 65% 5-year local control, but distant metastases became a prominent delayed failure pattern.

Specimen histology after one or two preoperative CF-252 implants for bulky stage IB cervical cancer.

Using hysterectomy specimens obtained 1 month after Cf-252 neutron brachytherapy plus fractionated radiotherapy, we determined the fraction of positive and negative specimens with neutron dose for bulky Stage IB cervical cancers. The specimens obtained and studied after an initial Cf-252 insertion when the sources were newer and less decayed were more frequently negative for histological evidence of cancer than after the sources had decayed and 2 insertions were needed. After two insertions to deliver a therapeutic dose preoperatively the specimens were more frequently positive. When a larger initial dose was delivered to the tumor a larger proportion of negative specimens was noted. The size of neutron dose fraction was important to local tumor clearance and to rendering the specimens negative as well as schedule in use.

Terminal deoxynucleotidyl transferase (TdT) in RadLV-induced C57BL thymomas.

Newborn C57BL mice exposed to RadLV at birth were followed and periodically sampled for thymus and bone marrow TdT activity. Tumors detected at or before 100 days had mostly low TdT levels. Large thymic lymphocytic tumors detected after 100 days had elevated total TdT content per gland. Serial samples showed a changing TdT pattern with time. The mid period shifted from low levels to normal-to-high levels for both TdT content and total TdT per gland. No unusual bone marrow TdT activity change preceded the appearance of thymomas. TdT marks a unique precursor cell highly sensitive to RadLV associated with lymphomagenesis. RadLV induced both TdT+ and TdT- thymic lymphomas and both types were found to occur after lymphoma initiation by injection of RadLV into newborns.

Adoptive transfer of immunity by spleen cells from LSA-lymphoma mice cured by BCNU.

BCNU treatment of advanced LSA lymphoma, a poorly antigenic tumor of syngeneic C57BL/ym mice, produced large numbers of cured mice which were highly immune against further LSA tumor challenges. Spleen cells from cured mice were transferred into normal naive mice in close temporal relationship to the injection of 10(3) LSA cells and showed that during a period of two days before to one day after tumor injection, the MST and per cent tumor takes could be greatly modified. Activity was only weakly present in bone marrow or thymus cells but mixtures of these cells with or without added spleen cells were effective. In 400 rad sublethally irradiated mice it was also found that transferred spleen cells prevented progression of tumor, and resistance was permanent. This indicates that all elements of tumor recognition, affector and effector limbs of immune response were present in the adoptively transferred spleen cell population.

Evidence for host resistance in 1,3 bis(2-chloroethyl)-1-nitrosourea treatment induced in syngeneic LSA lymphoma.

The anticancer agent 1,3 Bis(2-chloroethyl)-1-Nitrosourea (BCNU) cures the advanced syngeneic LSA lymphoma of C57BL mice with high efficiency. The cured animals resist further tumor challenge by large numbers of viable syngeneic tumor cells. Growth assays of spleen proliferation of the intravenously inoculated tumor revealed a progressive-regressive pattern of spleen growth after LSA-tumor injection. Lymphoma colony forming units (LCFU) in the spleen initially increased then regressed. In vitro assays of serum showed a lack of cytotoxic activity in mice cured by BCNU. Added spleen, thymus, or bone-marrow cells were similarly ineffective. Spleen and bone-marrow cells from immune mice passively transferred to normal mice showed weak cytotoxic activity against the LSA tumor. BCNU-cured mouse cells were more effective in protection than those cured with Chlorozotocin (CLZ).

Survival of cultured human cells exposed to californium 252Cf radiation at low dose rates.

Human T-1 cells were used to determine the rbe of low dose rate 252Cf (n + gamma) radiation, compared to low dose rate gamma radiation. The T-1 cells were irradiated hypoxically at room temperature, and the dose rates were as follows: a) 252Cf (n + gamma), 0.045, 0.090, and 0.270 Gy/hour; and b) 137Cs gamma radiation, 0.117, 0.246, and 0.765 Gy/hour. The RBE was obtained as the ratio of the initial slope of the 137Cs survival curves (approximately the same for all three dose rates), and the initial slope of the 252Cf survival curves (same for all three dose rates). The RBE was 5.0 +/- 1.0 for all components of the 252Cf radiation and 7.1 +/- 1.7 for the neutron components. As the dose increased, an inverse dose rate effect (more sensitive at lower dose rates) for the 137Cs survival curves was observed.

Schedule in Cf-252 neutron brachytherapy: complications after delayed implant therapy for cervical cancer in a phase II trial.

The objective of this study was to review severe complication frequency in a protocol study using a defined prescribed dose combined with fractionated whole pelvis radiotherapy to 40-45 Gy. The method used a dose of Cf neutrons to 35 Gy equivalents (relative biological effectiveness or RBE adjusted) to a total tumor dose of 80 Gy-eq in one to four implant sessions. Compliance was excellent, and most patients received two implants to 35 (0.4) (SE) Gy-eq in two sessions plus external radiation to a total point A or paracervical region dose of 80 (0.3) Gy-eqs. In patients who received delayed implants, the severe complication rate (pelvic necrosis, fistulas) was significantly greater (40% versus 3%). We postulate that neutron brachytherapy caused tumors to regress rapidly and completely, which allowed the neutron dose to adjacent radiosensitive organs (bladder, rectum, sigmoid colon, and bowel) to become excessive. The delayed Cf implant apparently contributed to the greater risk for normal tissue complications.

Five-year cure of cervical cancer treated using californium-252 neutron brachytherapy.

Female pelvic carcinoma is one of the common malignancies seen at the University of Kentucky Medical Center and often presents in an advanced stage. In 1976, we began to test californium-252 neutron brachytherapy (NT) for its efficacy for control of primary and recurrent advanced uterine, cervix, and vaginal cancers. The first protocol used was 5000-5500 rad of whole pelvis irradiation followed by 1-2 Cf-252 insertions using a single tandem placed in the utero-cervico-vaginal region. Of 27 patients with primary carcinomas treated, 10 are alive and well 5 years later (37%). Two of two recurrent tumors were locally controlled but failed later. These patients had advanced cervical, vaginal, or endometrial carcinomas. In 1977, a transitional year, treatment of only unfavorable stages and presentations with NT was initiated. Similar results were obtained with NT as compared to conventional photon therapy (PT). Further improvement in treatment results can be anticipated as NT brachytherapy is used for advanced cancer therapy by more effective treatment schedules and radiation doses. Cf-252 can be used as a radium substitute and achieved similar rates of tumor control and 5-year survivals.

Clinical study of relative biological effectiveness for cervical carcinoma treated by 252Cf neutrons and assessed by histological tumour eradication.

Clinical data of the University of Kentucky trial using californium (252Cf), or caesium (137Cs), are reviewed for dose-response based on the endpoint of tumour eradication estimated from hysterectomy specimens obtained 4-6 weeks after preoperative irradiation. These data are used to assess the relative biological effectiveness (RBE) for 252Cf neutrons compared with 137Cs gamma radiation. Tumours treated were of common stage but were of bulky or barrel shape suitable for "radiosurgical" therapy. Dose-response curves were constructed, and additional data from the literature used to analyse the curve shape. The photon dose-response curve is complex on a logarithmic plot, whereas the 252Cf neutron curve is exponential. This indicates that the RBE can be different depending on the number of implants, schedule and size of dose delivered per session. The RBE values were approximately 8.0 at low doses or for multiple implants but they may rise to approximately 16 at larger doses or for single 252Cf implants.