Victor Hofmann

Limitations of the salmonella/mammalian microsome assay (AMES test) to determine occupational exposure to cytostatic drugs

Urine samples of nursing personnel working in medical oncology divisions of several Swiss hospitals were examined for mutagenic activity. Urine samples were concentrated 100 times following XAD-2 chromatography and mutagenicity was determined using the Salmonella/mammalian microsome assay (Ames test). Apart from the urine samples of patients treated with cytostatic drugs and urine samples of nurses who are cigarette smokers, no mutagenic activity could be found. Also following exposure to an increased and defined quantity of cytostatic drugs no mutagenicity could be recovered from the urine. Four different nurses worked with cyclophosphamide, methotrexate, 5-fluorouracil, adriamycin and cis-platinum for 3-4 hr without using any protection such as gloves, masks or a vertical laminar airflow hood. Aqueous extracts of filters, through which air was pumped during the whole experiment (a personal air-sampler was fixed near the face of the test persons), were non-mutagenic. Parallel to the mutagenicity test chemical analyses were also done. The methotrexate content was determined in serum samples and the aqueous filter extracts and urine samples were examined for cis-platinum. All chemical determinations were negative. With the aid of urine concentrates of a patient treated with sub-therapeutic doses of cyclophosphamide as well as with normal urine to which single small amounts of different cytostatics (adriamycin, cyclophosphamide, cis-platinum) were added, the detection limits for the corresponding cytostatic drugs were determined and found to be in the range of 2-10 mg for cyclophosphamide and approx. 10 micrograms for adriamycin. Cis-platinum was lost during the passage through the XAD-2 columns. With these results at hand the sensitivity of the hitherto preferably used method (Ames test) for the monitoring of exposure to cytostatic drugs must be seriously questioned.

Effects of leukocyte interferon (E. coli) on human bone sarcoma growth in vitro and in the nude mouse

Effects of highly purified human leukocyte interferon (rIFN-alpha 2) on colony formation, DNA synthesis and proliferation in nude mice of tumor cells from eight bone sarcomas have been studied. rIFN-alpha 2 produced a dose-dependent inhibition of [3H]thymidine incorporation by sarcoma cells. Even at high doses (10(4) U/ml), however, [3H]thymidine uptake could not be completely blocked by rIFN-alpha 2. In a cloning assay three established sarcoma cell lines and five other sarcoma samples obtained after short-term in vitro culture were found to be sensitive to various degrees to rIFN-alpha 2, complete inhibition being seen only at 10(4) U/ml. Three sarcomas were sensitive in the nude mouse model. Scheduling experiments revealed that rIFN-alpha 2 produces a delay in tumor growth only when administered either before or shortly after tumor implantation. Therefore rIFN-alpha 2 appears to be most active when tumor size is small and growth not exponential, indicating that rIFN-alpha 2 may play a role in an adjuvant setting. Growth sarcomas strongly suppressed by rIFN-alpha 2 in the cloning assay was markedly inhibited in the nude mouse. One sarcoma which was only moderately sensitive in the cloning assay was resistant in the animal experiment, confirming the predictive value of the clonogenic assay. Although the present findings demonstrate strong antitumor activity of rIFN-alpha 2 against human bone sarcoma cells they should be interpreted with caution mainly because the high rIFN-alpha 2 levels used in the experiments cannot be maintained in patients over a prolonged period.

Predictive drug testing on human tumor cells

Clonogenic Assays for Solid Tumors.- In Vitro Growth of Human Malignancies in a Cloning Assay.- Development and Applications of a Human Tumor Colony Assay for Chemosensitivity Testing.- A Replenishable Soft Agar Colony Assay for Human Tumor Sensitivity Testing.- Analysis of Malignant Effusions by Cellular Composition, Proliferation Kinetics, and In Vitro Clonogenicity.- Direct Cloning of Human Ovarian Cancer in Soft Agar: Clinical Limitations and Pharmacologic Applications.- Technical Problems with Soft Agar Colony Formation Assays for In Vitro Chemotherapy Sensitivity Testing of Human Solid Tumors: Mayo Clinic Experience.- Cloning of Human Tumor Cells in Methylcellulose-Containing Medium.- The Effect of Chemotherapy on Human Bone Sarcomas: A Clinical and Experimental Study.- Predictive Tests for Hematological Malignancies.- Experimental Approaches to Outcome Prediction in Acute Myeloblastic Leukemia.- Experimental Approaches to Drug Testing and Clonogenic Growth: Results in Multiple Myeloma and Acute Myelogenous Leukemia.- In Vitro Assessment of Drug Sensitivity in Acute Nonlymphocytic Leukemia.- Short-Term In Vitro Sensitivity Testing in Acute Leukemia.- Non-Clonogenic Assays for Drug Testing.- Development of a Nucleotide Precursor Incorporation Assay for Testing Drug Sensitivity of Human Tumors.- Predictive Relevance for Clinical Outcome of In Vitro Sensitivity Evaluated Through Antimetabolic Assay.- Biochemical Short-Term Predictive Assay: Results of Correlative Trials in Comparison to Other Assays.- In Vitro Chemosensitivity Assay Based on the Concept of Total Tumor Cell Kill.- Evaluation of In Vitro Results.- Predictive Tests and Infrequent Events in Cancer Chemotherapy.- Pharmacologic Pitfalls in the Human Tumor Clonogenic Assay.- Heterogeneity and Variability of Test Results as Limiting Factors for Predictive Assays.- Interlaboratory Comparison of In Vitro Cloning of Fresh Human Tumor Cells from Malignant Effusions.- Pharmacology, Phase II Studies and New Drug Development.- Evaluation of Schedule Dependency of Anticancer Drugs in the Human Tumor Clonogenic Assay.- In Vitro Effect of Interferon-a on Human Granulocyte/Macrophage Progenitor Cells and Human Clonogenic Tumor Cells..- Effect of Leukocyte Interferons on Cell Proliferation of Human Tumors in Vitro.- Drug Combination Testing with In Vitro Clonal Cultures.- Neutralization of cis-Dichlorodiammineplatinum II and Nitrogen Mustard by Thiols.- Usefulness of the Human Tumor Colony Forming Assay for New Drug Development.- In Vitro Characterization of New Antiestrogens in Human Mammary Tumor Cells.- Modulation of Tumor Growth by Non-Chemotherapeutic Intervention.- Growth Factor Enhancement of the In Vitro Stem Cell Assay.- Improving Techniques for Clonogenic Assays.- Relationship of Steroid Hormone Receptors to the Cloning of Fresh Breast Cancer Tissues.

Hairy cell leukemia. Ultrastructural and cytochemical evaluation of leukemic colonies grown in a semi-solid medium☆

We examined the fine structure and enzymatic activity of cells composing colonies grown from blood and/or spleen of eight patients with hairy cell leukemia. Mononuclear cells (MNC) were plated over an agar layer in a medium containing methylcellulose and leukocyte-conditioned medium. After 7-10 days incubation colonies were harvested for cytologic study. Colony cells possessed a euchromatic nucleus with an occasional nucleolus. Their cytoplasm contained a prominent Golgi region, numerous mitochondria and small vesicles, many short strands of rough endoplasmic reticulum (RER) and an infrequent phagosome. Well-developed ribosome-lamella complexes and what may have been their intermediate forms appeared in colony cells from three patients. Strong activity for tartrate-resistant acid phosphatase, localized in the RER, nuclear envelope and some Golgi vesicles, was evident in 50-95% of all colony cells. Our results indicate that a high proportion of MNC forming colonies in this culture system maintain the characteristic morphology and cytochemical activity of hairy cells.

Effects of 13-cis retinoic acid and Ara-C on differentiation and proliferation of non-promyelocytic acute myelogenous leukemia☆

An alternative to a cell-kill strategy for eradication of acute myelogenous leukemia, is to restore normal differentiation. Vitamin A derivatives demonstrate differentiation-inducing activity both in vitro and in vivo on promyelocytic leukemic cells. We tested the ability of 13-cis retinoic acid to reduce proliferation and induce differentiation in 10 samples from patients with acute non-promyelocytic leukemia. DNA synthesis and leukemia colony formation were affected to varying degrees by a prolonged exposure to the vitamin A compound. Morphologically and cytochemically no differentiation was determined either after 48 h in suspension cultures or 7 additional days in semi-solid cultures. Alkaline leukocyte phosphatase, a biochemical marker of differentiation, was significantly increased in five samples. DNA synthesis in these samples was significantly reduced as compared to samples failing to express alkaline leukocyte phosphatase following 13-cis retinoic acid treatment. DNA synthesis of these same 5 samples was also strongly inhibited by Ara-C. Expression of alkaline leukocyte phosphatase following 13-cis retinoic acid exposure may be a useful indicator for cells amenable to 13-cis retinoic acid or Ara-C treatment.

Analysis of Malignant Effusions by Cellular Composition, Proliferation Kinetics, and In Vitro Clonogenicity

The human tumor clonogenic assay (HTCA) has received wide international attention and has been introduced in numerous laboratories since its original description by Hamburger and Salmon (1977). The dominant adavantage of the HTCA is its specificity for selecting neoplastic clones to the exclusion of other cellular elements. The neoplastic origin of cells comprising colonies has been confirmed by light and electron microscopy (Salmon and Liu 1979; Harris et al. 1982), production of tumor markers (Von Hoff et al. 1980), cytogenetics (Trent and Salmon 1980), and colony transplants into nude mice. Furthermore, it is believed that tumor clones growing in semisolid agar represent the cell fraction responsible for in vivo tumor propagation.

Prognostic significance of agar and liquid cultures in AML patients before treatment, early postinduction and in remission

In the present study, the growth and differentiation capacity of myeloid leukemic cells in agar and liquid cultures have been investigated in relation to their prognostic significance for treatment outcome and early detection of relapse. Prior to induction therapy, leukemic cells failed to differentiate and the colony or cluster number did not correlate with response to treatment. Seventeen to 42 days after induction, patients with BM cells producing greater than 10 colonies or greater than 30 clusters resp. had a high likelihood of achieving a complete remission. Cells from refractory patients had a significantly impaired differentiation capacity. During remission, a colony number greater than 50 was significantly associated with a high probability to remain in further remission for greater than 3 months. An impaired differentiation was significantly associated with the likelihood of relapsing within 3 months. In the light of these results, agar and liquid cultures appear to be useful for monitoring the effect of induction chemotherapy and detecting patients likely to relapse.

Leukemic samples characteristics associated with clonogenic growth

In this study, we attempted to delineate readily assessable characteristics from 100 leukemic samples associated with in-vitro growth. Successful growth defined as production of greater than 30 clusters and/or colonies per culture dish was obtained in 68% of samples. More than 10 colonies were found in 59% and greater than 30 colonies in 51% of cultures, respectively. Leukemic cells from patients previously treated with aggressive cytotoxic chemotherapy grew significantly better than cells from untreated patients, independently of the above definitions of cloning success. Cells from peripheral blood had a weak, albeit significant growth advantage over bone marrow cells (p = 0.032) when cluster growth was taken into account for growth success. When colony growth alone was used as criterium, no growth advantage was found. The morphological subtype and the proliferation kinetics prior to cell plating did not affect cloning success. A high labeling index had predictive value for subsequent growth, but only in bone marrow cells. By multivariate analysis, we found that treatment status was the most important factor correlated with in-vitro growth.

The Effect of Chemotherapy on Human Bone Sarcomas: A Clinical and Experimental Study

Osteosarcomas are relatively rare tumors affecting younger people, mostly men. The prognosis in these cases is uniformly bad. An analysis of the course of the disease 10 years ago revealed that roughly 10 months after removal of the primary tumor, lung metastases develop (Sweetnam et al. 1971). Before 1970, a 5-year survival of 20% or less is reported following surgical treatment of the bone tumor. Chemotherapy was essentially unsuccesful until the early 1970s, when trials with doxorubicin and high-dose methotrexate were initiated. With these two drugs, considerable response rates (mostly partial remissions) were reported in metastatic disease.

Clinical Experience with Preoperative Chemotherapy for Osteosarcoma

Since 1978 preoperative chemotherapy has been administered to 15 consecutive patients with osteosarcomas in Zurich. Preoperative chemotherapy was acceptably well tolerated and did not impair surgical procedures. Our retrospective analysis confirmed that the extent of necrosis after preoperative chemotherapy is of biological importance for the further course of the disease. Patients with extensive necrosis had better relapse-free survival and longer overall survival than those with little necrosis.