Temperature effect on protein synthesis in a heat-synchronized protozoan treated with actinomycin D.

Protein synthesis was studied in the ciliated protozoan Tetrahymena pyriformis GL after actinomycin D was added to the culture medium. When the temperature rose above that optimum for growth, there were significant reductions in protein synthesis. Lipid biosynthesis under the same conditions was slightly stimulated, an indication that the eflect was not due to an underlying reduction in energy sources. The phenomenon appears to be unique to the protein synthesizing system. Correlation with previous data suggests that it is due to nontranslational destruction of template RNA.

A comparison of the acute effects of radiation therapy, including or excluding the thymus, on the lymphocyte subpopulations of cancer patients.

Radiation therapy to either mediastinum or pelvis causes a rapid decrease in circulating lymphocytes of both B and T types and in addition an impairment in the function of the remaining lyphocytes, as measured by their ability to proliferate in response to mitogens. The acute depression is short-lived. Substantial recovery is apparent within 3 wk after cessation of therapy; however, most patients show a modest, chronic depression in both numbers and functional capacities of circulating lymphocytes. T cells are somewhat more sensitive than B cells, but both are affected. Irradiation of the thymus per se seems to have little influence on the acute changes which occur, as patients receiving pelvic and mediastinal (including thymic) radiotherapy show a similiar degree of lymphopenia and depression of lymphocyte responsiveness.

Induction of DNA degradation in vivo by adriamycin.

The anthracycline antibiotic adriamycin (Ad) induced degradation (single-strand breaks) of cell DNA in vivo in murine L1210 leukemia cells. Measurement was made by alkaline sucrose gradient methods. Exposure of isolated DNA to Ad had little effect; the in vivo DNA strand breaks were probably due to the action of nucleases on DNA distorted by bound Ad. Toxicity of Ad to postmitotic cells such as cardiac cells could be mediated by this mechanism, since it could be demonstrated in periods substantially shorter than the cell generation time and thus could be responsible for interphase death.

Molecular interactions of the combined effects of bleomycin and x-rays on mammalian cell survival.

The interactions between bleomycin and X-ray damage and repair have been examined in rat and human tumor cells. Bleomycin itself indices extensive DNA single-strand breaks but does not appear to inhibit the repair of X-ray-induced DNA single-strand breaks. Quantitative analysis of these interactions is complicated by the retention of active bleomycin within cells that remains capable of further DNA degradation even under the conditions of alkaline sucrose gradient cell lysis. DNA double-strand breaks and/or disruptions of DNA-lipid complexes also occur following bleomycin exposure. X-ray-induced excision repair replication is only minimally influenced by even high concentrations of bleomycin. A small amount of excision repair is demonstrable in nonirradiated cells treated with high concentrations of bleomycin consistent with repair of bleomycin-induced nucleotide damage in cellular DNA by a "cut and patch" repair mechanism. Repair of bleomycin-induced DNA single-strand breaks also occurs. The data indicate that bleomycin and X-ray damage are quite similar both in their induction and repair, but that lesions occur and are repaired independently. The enzymatic mechanisms appear similar in the two cell types despite substantial differences in their sensitivity to bleomycin.

Phase I and II trial of five-day infused 5-fluorouracil and radiation in advanced cancer of the head and neck.

Eighteen patients with advanced epithelial cancers of the head and neck region were studied for their tolerance and response to combined cycles of 120-hour infused 5-fluorouracil (5-FU) and external-beam radiation therapy. 5-FU infusions were given under conditions where radiosensitization would be expected at the higher infusion doses. Coincident radiation treatments were given as four sequential daily fractions of 250 rad each administered during days 1 through 4 of each five-day infusion cycle. The patients were rested for at least nine days after each cycle or longer until toxicity was resolved. The regimen was then repeated in each patient for a total of five treatment cycles. Thereafter therapy was consolidated, usually by boost radiation without drug. In sequential patient subsets the infusion load was progressively escalated in a phase I format. The complete response rate for stage IV patients was 75% with survival benefit compared to prior results. 5-FU dose-dependent combined modality loco-regional toxicity was demonstrated without significant enhancement of systemic toxicity of any form; 5-FU dose-dependent enhanced responsiveness and survival benefit is also suggested. Further scheduling and response studies of 5-FU under radiosensitizing conditions appear warranted.

Thermal marrow expansion: effect of temperature on bone marrow distribution.

The distribution of bone marrow in adult mammals is limited mainly to those bones deeply embedded in the body and subject to the higher core body temperature. Active marrow is much less prevalent in the limbs. In the past this was attributed to the lower temperature of the peripheral bone marrow. We have studied the possibility of stimulating hematopoiesis in the limbs (and tails) by increasing the limb temperature. This has been done by elevating the ambient temperature of mice and by tail implantation techniques. Mice incubated at ambient temperature of 22 degrees C can be "expanded" to have markedly enhanced marrow activity in the limbs by either incubation for seven days or more at 34 degrees C, or by implantation of the bone into core temperature areas (peritoneal implantation). The increase in marrow activity is shown both histologically and by 59Fe uptake techniques. Anemia does not enhance this effect, indicating a local phenomenon. A variety of experiments suggest that temperature per se and not temperature-induced increase in blood flow is involved. The increase in marrow activity is accompanied by a modest local increase in clonogenic marrow stem cells but it cannot be shown for certainty whether the phenomenon of Thermal Marrow Expansion (TME) is due to the local effect of elevated temperature on stem or stromal cells. The erythrocytes produced by TME appear normal by in vivo fragility testing. TME may have clinical usefulness in man if the phenomenon occurs in the same fashion as rodents.

Exclusion of an interactive effect of combined x-irradiation and activated cyclophosphamide in tissue culture.

The effect of Cyclophosphamide (CY) on the X ray survival of clonogenic tumor cells has been studied in vitro. Two activated derivatives of the drug, Peroxycyclophosphamide and Hydroperoxycyclophosphamide, were employed. Two cell lines, repair-competent human HeLa cells and the repair-deficient rat REQ line, were investigated. Neither form of CY had any effect on the X ray survival curve of either cell line, indicating that any interaction anticipated in vivo could be expected to be additive.

Enhanced accumulation of 5-fluorouracil in human tumors in athymic mice by co-administration of Ftorafur and uracil.

Human colonic tumors grown in athymic mice were tested for the effect of coincident uracil (U) and Ftorafur (FT) exposure versus FT alone on 5-Fluorouracil (5-FU) metabolism. Serum and tumor FT and 5-FU levels were studied as a function of time after FT +/- U injections. The combination of U + FT led to significantly higher tumor/serum 5-FU ratios than FT alone. The data indicate that the metabolism of 5-FU released from FT can be modulated by coincident U exposure in human tumor cells in vivo. Such combinations may be of use in the development of oral 5-FU pro-drugs for applications using 5-FU as an out-patient non-invasive radiosensitizer.

Pharmacologic requirements for obtaining sensitization of human tumor cells in vitro to combined 5-Fluorouracil or ftorafur and X rays.

The combined effects of X ray and 5-Fluorouracil (5-FU) in tissue culture have been studied using two human adenocarcinoma lines (HeLa and HT-29 cells). Both showed similar sensitivities to 5-FU, HeLa cells appearing somewhat more resistant to higher concentrations. Combined treatment of both cell types with 5-FU and X rays led to a time-dependent enhancement of cell killing ("radiosensitization"). Only post-radiation incubation had any effect, prior exposure to 5-FU being strictly additive. Enhanced cell killing by combined 5-FU and X rays could not be explained by either the infliction of additional acute damage in the immediate post-radiation period or an inhibitory effect of 5-FU on the repair of sub-lethal X ray injury. Rather, the enhanced cytotoxicity proved to be dependent on a damage expressed in time periods exceeding the duration of a cell doubling time in vitro. Overall, the data equally suggest that X rays may sensitize the cells to 5-FU. The enhanced cell killing is maximized if the cells are continuously exposed to 5-FU for 48 hours following the X ray exposure. These results indicate that clinical treatment regimens might be useful in evaluating 5-FU infusional scheduling in accordance with these unique requirements, which are not met by conventional bolus 5-FU and X ray therapy fractionation regimens. Ftorafur, a drug proposed to act as a slow release form of 5-FU, was found to show limited cytotoxic potential in vitro and did not significantly enhance cell killing after X ray exposure.

Phase I and pharmacologic study of 72-hour infused 5-fluorouracil and hyperfractionated cyclical radiation.

We have studied 21 patients infused for 72 hours with 5-Fluorouracil (5-FU) at progressive doses combined with hyperfractionated radiation. The schedule was chosen as being one capable of inducing 5-FU radiosensitization (RS). All patients were started at a daily 5-FU dose of 40 mg/kg/24 hours; doses were then escalated with each subsequent treatment cycle to limiting toxicity or until taken off study. Patients received between one and six infusion cycles. Every treatment cycle included coincident hyperfractionated radiation to various body areas including the abdomen, chest, and head and neck region. Radiation fractionation was invariant; 1,000 rad were delivered in four equal fractions. Two fractions of 250 rad each were given on days 1 and 2 of each three day 5-FU cycle, i.e. at approximately 0, 8, 24, and 32 hours into the drug infusion. Patients were followed for toxicity; serum 5-FU concentrations were determined using a high pressure liquid chromatographic assay. 5-FU clearances were calculated from the mean serum drug levels and the infused drug dose. The toxicity spectrum was not found to be significantly different from infused drug alone in this dose range save when the head and neck region received coincident irradiation. In that region the two anticipated toxicities combined in what appears to be a synergistic fashion to enhance mucositis. Most toxicities including gastrointestinal and bone marrow appeared dependent on the mean serum 5-FU level as did mucositis itself. 5-FU clearance was found to be non-linear in this dose region but did not appear influenced by radiation to any part of the body. This study shows that 72-hour infused 5-FU can be combined with external beam radiation and will produce reasonably predictable toxicity patterns which depend on the region of the body being irradiated. 5-FU toxicity correlates with mean serum drug level which is itself dependent on 5-FU clearance. Minor variations in 5-FU clearance therefore probably contribute to the natural range found in the dose-response relationship for infused 5-FU toxicities. Future studies should integrate this understanding of 5-FU pharmacokinetics into treatment regimens. The combination of infused 5-FU and coincident radiation appears useful in treating several tumor types, particularly squamous and squamous-like cancers. However, further scheduling and radiation fractionation studies are desirable to optimize 5-FU RS in man and to quantify late effects.

Bleomycin, cyclophosphamide and radiotherapy in regionally advanced epidermoid carcinoma of the head and neck.

We have treated 24 patients with squamous carcinoma of the head and neck and advanced regional (N2-3) disease. The regimen consisted of 3 cycles, each of 28 days. Cyclophosphamide (1 gm/m2 I.V.) was given on day 1, bleomycin (15 u I.M.) on days 2, 4, 9 and 11, and ionizing radiation (60Co, 180 rad/fraction) days 1-5, and 8-12. No therapy was given on days 13-28. After three cycles of therapy, 13 patients had a complete response; following further therapy (surgery, interstitial or external beam radiation), 16 patients were free of disease. However, remissions were not durable and 11/16 patients recurred loco-regionally with a median time to recurrence of 5 months; most (7/11 also developed distant metastases. These patients have biologically aggressive disease and may have a worse prognosis than patients who are Stage IV based on a T4 primary lesion only.

Phase I and pharmacologic study of oral ftorafur and X ray therapy in advanced gastrointestinal cancer.

We have treated 15 patients with advanced gastrointestinal carcinoma with a cyclical regimen of combined Ftorafur (N1-((2-furanidyl-))-5-Fluorouracil, a 5-FU pro-drug) and external beam radiation. The Ftorafur (FT) was administered orally in daily doses of between 1.0 and 2.5 g/m2/day in 3 divided doses in a Phase I format. The drug was given daily for 5 days along with conventional X ray treatment portals and daily radiation doses of 250 rad on each of the first 4 days of each treatment cycle. The patients were then rested for a minimum of 10 days or until all significant side effects had passed. The total number of 1,000 rad cycles and radiation dose were dictated by tolerance and by normal organ dose limitations. The most common toxicity in general, and the most common limiting toxicity was nausea and vomiting, in contrast to oral FT alone where diarrhea is more prominent. Stomatitis was seen only once and no other form of serious toxicity was encountered. Two-thirds of the patients responded in subjective terms (pain relief). There was 1 partial response to FT alone (pulmonary metastases outside the treatment field). The sole patient whose treatment field was outside the abdomen (chest portals for esophageal carcinoma) developed pneumonitis which contributed to his death. No other delayed effects were noted. Serum FT levels were related to the ingested dose and in the microgram range while serum 5-FU levels were in the nanogram range indicating slow decomposition of FT into 5-FU. The therapy was reasonably well tolerated at doses of 2.0 g/m2/day or lower with abdominal radiation. FT offers the potential for replacing intra-venous infused 5-FU as a clinical radiosensitizer.

Interstitial-implant radiotherapy in upper-aerodigestive-tract malignancy.

In the treatment of upper-aerodigestive-tract malignancy, interstitial-implant radiotherapy differs in several physical and biologic respects from conventional external-beam therapy: unlike external-beam therapy, it can deliver a high central dose with a rapid fall-off, which overcomes the central hypoxic resistance effect and yet greatly improves normal tissue tolerance. External-beam and interstitial-implant therapy can also be combined to the patient's benefit; in many cases, this combination offers advantages over external-beam therapy alone, preoperative external-beam therapy, or aggressive surgery. The recent upsurge of interest in this modality has come about because of three developments: more suitable radioactive sources, after-loading, and computerized dosimetry. The early results in upper-aerodigestive-tract malignancy, both as primary and as salvage therapy, are promising; but the precise role of this treatment in our therapeutic armamentarium remains to be established.

Radiation therapy, local tumor control, and prognosis in bronchogenic carcinoma: current status and future prospects.

While the overall prognosis for cure of bronchogenic carcinoma remains poor for most patients, there is a growing body of evidence suggesting that rationally optimized local therapy may benefit a significant subset of patients. Local therapy in this context includes any systemic therapy (such as chemotherapy or immunotherapy) that enhances local tumor control in the chest. Compared with many other human epithelial cancers, the total local tumor burden is large for many nonresectable lung cancers and not within the tolerance for control by radiation alone. Thus there is growing evidence that combined surgery and radiation treatment will improve results, especially in the differentiated tumors. Proper selection of patients is important and must include histologic stratification in addition to conventional TNM staging. It is projected that much useful research can be conducted during this decade using clinical tools now available and those that are being tested in early clinical trials throughout the world. Likely candidates for such improvements are both oxic and hypoxic radiosensitizing drugs that should decrease the death rate from uncontrolled local cancer in the chest.

Irreparable DNA cross-links and mammalian cell lethality with bifunctional alkylating agents.

The appearance and removal of DNA cross-links in C3H10T1/2 cells has been investigated with closely related bifunctional alkylating agents having widely different kinetics of alkylating activity. Concentrations of nitrogen mustard, phosphoramide mustard and melphalan demonstrating equivalent levels of cell lethality indicate a correlation between the appearance of residual DNA cross-links, transformation and the loss of colony formation ability. Residual DNA cross-links appear to remain after repair is nearly complete by 24 h and consist of both the interstrand DNA and DNA-protein varieties. These adducts occur in the general population of cells despite the fact that these cells retain their DNA cross-link repair capabilities. Less than 100 interstrand DNA cross-links and a 10-fold greater number of DNA-protein cross-links are estimated to remain in the DNA after a 66% lethal dose of these compounds. Due to the obvious complications involving DNA replication in the presence of such lesions, residual DNA cross-links may possibly be associated with lethality in these cells. Current evidence demonstrating the formation of additional sites of alkylation such as irreparable phosphotriester adducts by several alkylating agents may suggest a role for a specific type of DNA damage in this process.

Improved liquid chromatographic assay for serum fluorouracil concentrations in the presence of ftorafur.

An improved liquid chromatographic assay for serum ftorafur and fluorouracil is shown to be routine, sensitive, and reproducible using 200 micro l of serum. Dilute ammonium acetate buffer at pH 10.2 is used for solubilization of the evaporated ethyl acetate extract for injection into the liquid chromatograph. A stability study indicated little or no in vitro formation of fluorouracil from ftorafur under the conditions described. Low serum fluorouracil levels were found after administration of therapeutic doses of ftorafur.

Bleomycin: its utilization in the treatment of head and neck cancer.

Originally isolated as an antibiotic, bleomycin is a complex mixture of glycopeptides currently utilized against a variety of malignancies, among them epidermoid, cancer of the head and neck. Because of unique independent pharmacological properties, bleomycin may be effectively used at several points in the natural history of tumors. Use of bleomycin as a single agent, as an adjunct to surgery and irradiation and as a palliative drug in head and neck cancer, is now advocated in scattered reports. The rationale for the utilization of bleomycin at the University of California, San Diego and San Diego Veterans Administration Hospitals is discussed. In addition, examples from the authors' personal series of both response and adverse effects, suggestions for appropriate clinical monitoring to detect early toxicity, and tennets for management of side effects are included in the presentation.

Hematologic parameters in the adjustment of chemotherapy doses in combined modality treatments involving radiation.

The differential white blood cell count of a group of patients with Stages I and II infiltrating ductal carcinoma who underwent treatment in the preadjuvant chemotherapy era have been evaluated. All patients received a modified radical mastectomy followed by postoperative radiation therapy to the chest wall and draining regional lymph node chains (ipsilateral internal mammary, axillary,and supraclavicular regions). When the levels of circulating neutrophils, band cells, and lymphocytes were compared for the period beginning prior to surgery and ending 1 year after the completion of radiotherapy, it was found that radiation induced a significant lymphopenia. However, all patients maintained a neutrophil count at least twice that needed for full-dose conventional chemotherapy. Based on these observations and related preclinical and clinical information, it is proposed that future clinical trials utilizing even local radiotherapy as a component of therapy must have their chemotherapy doses based on appropriate hematologic parameters (neutrophil + band count) in order to avoid spurious and quite possibly erroneous results.

Treatment with combined intra-arterial 5-FUdR infusion and whole-liver radiation for colon carcinoma metastatic to the liver. Preliminary results.

Twenty-eight patients with colon carcinoma metastatic to the liver were treated with continuously infused intra-arterial 5-fluorouracil deoxyriboside (5-FUdR) and cyclical whole-liver radiation (2000-3000 rad). Survivorship ranged from 25 days to almost 4 years and was a clear function of the extent of liver dysfunction at the time of initiation of this treatment. Difficulties in establishing the objective complete response rates in patients with minor imaging abnormalities were frequently noted. Both extracorporeal and permanently implanted arterial infusion devices have been employed, the results favoring the internal infusion units. Under ideal circumstances (early treatment, disease limited to the liver, and a permanent indwelling pump), a median survivorship of approximately 2 years can be projected with a significant number of patients rendered free of progressive cancer in the liver for months to years. The dose-limiting feature of this approach is treatment-related to hepatitis, which proved lethal in one of 28 patients thus far treated. Preclinical studies on the original and reduction of drug- and x-ray-induced liver toxicity should have high research priority.

Phase I and pharmacologic study of 72-hour infused 5-fluorouracil in man.

The relationships between the administered dose, clearance, and the toxicity spectrum of 5-fluorouracil (5-FU) administered as 72-hour constant infusion have been studied in 21 patients with advanced cancer. This was done as a pilot study for possible future combination using 5-FU as a radiosensitizer. Individual patients tolerated up to 65 mg/kg/24 hours, but serious toxicity appeared once as low as 35 mg/kg. Limiting toxicity proved to be "mixed" with upper intestinal symptoms (nausea and vomiting), stomatitis, and central nervous system signs all occurring in various patients. Central nervous system effects (both cerebellar and vomiting) proved as troublesome as stomatitis. There was only a general link between the administered dose and the subsequent toxicity grade, but a reasonably quantitative relationship emerged when the serum 5-Fu levels obtained and the degree of patient toxicity were compared. The clearance of 5-FU was confirmed to be nonlinear over the entire dose range studied (25-65 mg/kg/24 hours), consistent with a two-compartment model of drug metabolism. One compartment appears to be systemic (extra-hepatic) metabolism (probably anabolic removal) which is saturated at just below 15 mg/kg/day. Doses above that level lead to drug accumulation. No steady state was reached, contrary to previous reports. At the higher infusion rates, clearance progressively approaches that predicted by the assumption that the second compartment is splanchnic blood flow and catabolism. While 5-FU can be administered as a 72-hour infusion as one possible schedule for use as a single agent or for combined modality studies, CNS effects are quite troublesome in comparison to longer infusions to toxicity.