Daniel Glaubiger

Qualitative and quantitative aspects of intercalator-induced DNA strand breaks.

The intercalating agents, adriamycin and ellipticine, were previously found to produce DNA strand breaks associated with DNA-protein crosslinks in mouse leukemia L1210 cells. The current work explores the nature of the agents that produce this effect and the quantitative relationship between the breaks and crosslinks. The protein-associated DNA breaks were produced by a wide variety of intercalators in addition to the above-mentioned compounds: actinomycin D, daunoycin, ethidium and lucanthone (miracil D). Treatment with several drugs that bind to DNA without intercalation, or that inhibit DNA synthesis without binding to DNA, did not cause DNA breaks. The strand break and crosslink frequencies were quantitated by means of alkaline elution methods. The strand break and crosslink frequencies were found to be within a factor of 2 of each other over a range of concentrations of adriamycin and ellipticine. It is proposed that intercalation-induced distortion of the DNA helix leads to strand scission by a nuclease which becomes bound to one terminus of the break so as to form a DNA-protein crosslink.

Protein-associated DNA breaks in cells treated with adriamycin or ellipticine.

The effect of intercalating agents on mammalian DNA in vivo was examined by the technique of alkaline elution. Adriamycin and ellipticine were found to produce large numbers of single-strand breaks. These breaks appeared to be intimately associated with protein to the extent that enzymatic deproteinization of the DNA was necessary to reveal the breaks. The frequency of breaks and cross-links increased with concentration and time of exposure to the drugs. These data suggest that DNA single-strand scission may be a feature common to intercalators. The association with a cellular protein is previously undescribed and suggests possible mechanisms for the strand scission.

Duration of empiric antibiotic therapy in granulocytopenic patients with cancer

Abstract Early initiation of empiric antibiotic therapy in febrile cancer patients has become established practice, but the appropriate duration of antibiotic therapy when no infectious source can be identified is unknown. The complications of broad-spectrum antibiotics argue for brief treatment, but the risk of an inadequately treated infection in the granulocytopenic patient favors longer therapy. We prospectively studied 306 episodes of fever and granulocytopenia in 143 patients with leukemia or solid tumor (age one to 33 years) with respect to the duration of empiric antibiotic treatment. Eligible patients (fever > 38 °C three times/24 hours or > 38.5 °C once, plus polymorphonuclear leukocytes 3 ) had an extensive diagnostic evaluation, including at least two preantibiotic blood cultures, and therapy was then started with a broad-spectrum antibiotic regimen— Keflin ® , gentamicin and carbenicillin (KGC). Initial evaluation failed to identify an infectious etiology for the fever in 142 of 306 (46 per cent) episodes. Fifty-six of 142 (39 per cent) of these fevers of unknown origin were associated with persistent granulocytopenia for more than seven days; in 33 of these, defervescence occurred while the patients received KGC. After seven days of empiric KGC therapy, the 33 patients with fevers of unknown origin who had become afebrile with empiric antibiotics but whose polymorphonuclear leukocytes remained less than 500/mm 3 were randomized to either continue or discontinue (dc) to receive KGC. The patients who continued to receive KGC until their polymorphonuclear leukocytes were more than 500/mm 3 had no infectious sequelae. However, in seven of 17 (41 per cent) of the patients randomized to dc KGC infectious sequelae developed (p = 0.007) within a median of two days of discontinuing KGC (two with fever which again responded to KGC therapy, and five with a documented infection [two ultimately fatal]). In none of the patients did a resistant microbial flora or superinfection develop. These data suggest that the patient with a fever of unknown origin who becomes afebrile during empiric antibiotic therapy may profit from continued therapy while granulocytopenia persists.

Salvage irradiation of oropharyngeal cancers using iridium 192 wire implants: 5-Year results of 70 cases

Between May 1971 and November 1980, 70 patients with recurrent or new oropharyngeal cancers arising in previously irradiated tissues were treated using iridium 192 afterloading techniques. The actuarial local control was 72% at 2 years and 69% at 5 years. Although local control of the tumor was achieved in the majority of these patients, only 10 patients remained alive at 5 years (14%). Patients with lesions of the faucial arch and posterior pharyngeal wall had the best results; local control was achieved in 100% of these patients. Patients with lesions of the base of tongue and of the glosso-tonsillar sulcus had poorer results; local control was achieved in 61%. Because these results compare favorably with the results of previously published series, we recommend re-irradiation with brachytherapy for recurrent or new malignancies arising in a previously irradiated oropharynx. When the lesion is located in the faucial arch, brachytherapy is the treatment of choice. When the lesion is located in the base of tongue, brachytherapy is a reasonable option.

Determination of prognostic factors and their influence on therapeutic results in patients with Ewing's sarcoma

We have analyzed the results of treatment of 117 patients with Ewings sarcoma admitted to the National Cancer Institute since 1964. All patients received local irradiation to the primary site and a series of progressively more intensive systemic chemotherapy regimens using drugs known to be active as single agents in this disease. Four protocols were employed with varying numbers of patients in each treatment group. Initially, there appeared to be a difference among treatment groups with regard to disease‐free survival (overall P =.06), with the later regimens having more favorable outcomes. We then undertook a statistical analysis of the influence of five pretreatment variables—age, sex, site of primary disease, serum lactic acid dehydrogenase (LDH), and metastatic status—on disease‐free survival. Of these five factors, important indicators of favorable prognosis for the entire group (and for each of the treatment subgroups) were a distal site of primary disease, normal serum lactic acid dehydrogenase (LDH) level at presentation, and the absence of metastatic disease at the time of presentation. When we examined treatment results with respect to these prognostic factors, we found that the subgroups treated with the more aggressive regimens contained higher proportions of patients with favorable prognostic factors. After adjustment for differences in composition of treatment groups with respect to prognostic factors, the apparent difference in disease‐free survival vanished (P =.62). These results indicate that in the case of Ewings sarcoma, prognostic factors must be carefully considered in the design of treatment protocols and the subsequent analysis of end results.

Extraskeletal Ewing's sarcoma: results of combined modality treatment

Eleven patients with extraskeletal Ewings sarcoma (EES) were treated with combined modality therapy at the National Cancer Institute. The diagnosis of EES was reserved for lesions that were identical to Ewings sarcoma of bone by light and electron microscopy. Diagnostic work-up to rule out a skeletal primary included bone scan, localized views of adjacent bone, and bone tomography. Seven patients presented with an extremity primary and four patients had a truncal primary. No patients had evidence of metastases at presentation. Patients were treated with combined modality therapy consisting of high-dose local irradiation and vincristine, actinomycin D, and cyclophosphamide chemotherapy following a biopsy or local excision. No attempt was made to excise widely the primary tumor mass. Gross tumors generally responded rapidly to the combined modality treatment. Of 11 patients, seven (64%) remain disease free, with a follow-up of three to seven years from completion of therapy. Long-term local control was established in nine of 11 patients (82%). Autopsy findings on two patients with local failure showed no tumor involvement of adjacent bone. Attempts at gross resections by radical surgical procedures do not routinely appear to be necessary in light of the high local control rates with high-dose irradiation.

The reaction of anthramycin with DNA. II. Studies of kinetics and mechanism.

Abstract Anthramycin is known to react firmly with DNA, but no chemical basis for this interaction has been elucidated. Since anthramycin readily undergoes hydrolytic changes at C-11, the possible relation of this reaction to the interaction with DNA was considered. Both the hydrolysis of an 11-methoxy group and the reaction with DNA were found to be H+ catalysed, thus supporting a relation between the two processes. The titration of H+ from the phenolic group at Position 9 of anthramycin was found to be absent in the DNA complex, suggesting that Position 9 also is involved in the complex. The rate of reaction of anthramycin-11-methyl ether with DNA was found to be increased by prior hydrolysis. The rate of reaction with DNA, however, does not involve a rate-limiting conversion of anthramycin to a reactive component, since the kinetics of the reaction were found to be basically bimolecular. This excluded the possibility that a slow conformational change of the DNA was rate-limiting. The findings support the proposal that the binding of anthramycin to DNA is covalent, and that Positions 11 and 9 are involved in the binding. The bimolecular rate constant was found to decrease exponentially with the extent of DNA reaction, probably due to neighboring-site exclusion effects. The site of reaction involves guanine, since only guanine-containing deoxypolynucleotides were found to be reactive, and since reaction of DNA with anthramycin caused stochiometric elimination of binding sites of actinomycin.

Haemopoietic recovery in Ewing's sarcoma after intensive combination therapy and autologous marrow infusion.

After the completion of combination therapy designed to achieve local control of Ewings sarcoma, 13 patients with either truncal primary lesions or proven metastases were given 150 rad of total body irradiation over 5 weeks followed by cyclophosphamide, doxorubicin, imidazole carboxamide, and vincristine. 11 patients received autologous cryopreserved marrow infusions. In 2 patients marrow collections were not attempted. Two patterns of haemopoietic recovery were observed: 8 patients, who had received marrow infusions, showed leucocyte, granulocyte, and platelet recovery by 27, 28, and 30 days. 5 patients, 3 of whom had also received marrow, showed more delayed recovery with leucocyt, granulocyte, and platelet recovery at 45, 53, and 77+ days. Delayed recovery in patients receiving marrow seemed to correlate with aberrations in marrow freezing-rate during phase change, and these aberrations could be shown to diminish post-freeze recovery of marrow granulocyte-monocyte precursor cells.

Actuarial risk of isolated cns involvement in Ewing's sarcoma following prophylactic cranial irradiation and intrathecal methotrexate

Records of 154 patients with Ewings sarcoma treated at the National Cancer Institute were reviewed to assess the incidence and risk of developing isolated central nervous system (CNS) Ewings sarcoma. Sixty-two of the 154 patients had received CNS irradiation and intrathecal (i.t.) methotrexate as part of their initial therapy to prevent the occurrence of isolated CNS Ewings sarcoma. The risk of developing isolated CNS Ewings sarcoma was greatest within the first two years after diagnosis and was approximately 10%. The overall risk of CNS recurrence in the group of patients receiving CNS treatment was similar to the group receiving no therapy directed to the CNS. The occurrence of isolated CNS involvement was not prevented by the use of CNS irradiation and i.t. methotrexate. Because of a lack of efficacy to the CNS irradiation regimen, current treatment regimens do not include therapy directed to the CNS.

The reaction of anthramycin with DNA: III. Properties of the complex

Abstract Anthramycin has a high degree of specificity for reaction with helical DNA. This specificity must reflect some special configurational relationship between anthramycin and helical DNA. Several physical properties reflecting configuration of anthramycin—DNA complexes were studied. Intrinsic viscosity and radius of gyration were found to be increased in the complex, but only at high anthramycin DNA ratio, and only at sufficiently high DNA molecular weight. There was no significant change in sedimentation constant. These findings argue that at high extents of binding, the DNA helix becomes stiffened but not lengthened. Electric dichroism measurements indicated that the chromophore portion of the anthramycin molecule is oriented at an angle of approx. 36° relative to the helix axis. Anthramycin therefore is not intercalated between base-pairs. Circular dichroism measurements supported the suggestion that anthramycin in the complex resembles the deprotonated form. The patterns for complexes with native and denatured DNA were basically similar. From this and previous data, it is suggested that the major binding of an anthramycin molecule with helical DNA involves only one strand.