The genetic origin of drug resistance in neoplasms: implications for systemic therapy.

Drug resistance continues to be a major factor in limiting the effectiveness of cancer chemotherapy. Evidence from a variety of sources implicates a genetic basis for most drug-resistant phenotypes. Assuming a random spontaneous origin for these resistant cells, it is possible to develop mathematical and computer-based models of the drug treatment of tumors. These can provide a more intuitive understanding of the basis of treatment success or failure. This in turn may lead to the development of more rational and effective treatment protocols. Studies of phenomena such as pleiotropic drug resistance are providing insights into how multiple levels of drug resistance occur and are yielding information on how certain types of drug resistance may be prevented or overcome.

Heterogeneity in the specific activity and methotrexate sensitivity of dihydrofolate reductase from blast cells of acute myelogenous leukemia patients.

Dihydrofolate reductase activity was found to be highly heterogeneous in terms of its specific activity and methotrexate sensitivity in the blast cells of patients with acute myelogenous leukemia. None of the patients had previously been treated with methotrexate (MTX). The blast cells of four of 12 patients studied contained methotrexate-insensitive forms of dihydrofolate reductase, and the blast cells of three (distinct from the four mentioned previously) of the 12 had significantly higher dihydrofolate reductase activities than the rest. The presence of MTX-insensitive dihydrofolate reductases and high levels of enzyme activity represent intrinsic mechanisms of resistance and may explain the apparent clinical resistance of acute myelogenous leukemia to methotrexate.

Eradication of human non-Hodgkin's lymphoma in SCID mice by BCL-2 antisense oligonucleotides combined with low-dose cyclophosphamide.

Cancers overexpressing Bcl-2 protein, which prevents programmed cell death (apoptosis), are less sensitive to stresses that produce cellular damage, including chemotherapy. If the level of Bcl-2 protein can be reduced sufficiently using antisense oligonucleotides (ASOs) targeting the gene message, then cytotoxic agents may be rendered more effective in eliminating disease and increasing cure rate. Preclinical studies in SCID mice bearing Bcl-2 overexpressing systemic human B-cell lymphoma (DoHH2) were undertaken to support development of a clinical trial. These data confirm that a combination of an ASO (5 mg/kg) targeting bcl-2 and a low dose of cyclophosphamide (35 mg/kg) was an effective strategy, leading to the eradication of the DoHH2 cells in vivo and cure of the animals. When mice deficient in natural killer cell activity were treated with an ASO, similar results were observed, suggesting that ASO stimulation of the host immune system was not a significant factor in elimination of lymphoma cells. These studies indicate that therapeutic strategies involving the use of an ASO targeting bcl-2 in combination with a cytotoxic agent may improve clinical outcomes.

Scientific basis for adjuvant and primary (neoadjuvant) chemotherapy.

It can be stated as a general biological principle that there are many compelling reasons why chemotherapy should be directed at minimal tumor burdens. This is true whatever the nature of the tumor and becomes especially valid when one is dealing with tumors that are not curable when treated at the advanced stage. The patients who are likely to have the greatest benefit from adjuvant chemotherapy are, somewhat paradoxically, those who are at the least risk for recurrence following primary treatment. This is because, on the average, these patients will have the least tumor burdens. Patients who are at very high risk for relapse in breast cancer, (stage II patients with four plus positive nodes) will be the ones with the greatest subclinical burdens and may well have already crossed the threshold of curability to incurability. Directing effective chemotherapy programs at patients with lesser risk of recurrence complicates the ethical problems associated with adjuvant chemotherapy. To some degree, these ethical concerns can be assuaged by the appreciation that it is likely that protracted programs of chemotherapy (1 to 2 years) may well not be necessary. In general, curative drug programs can generally accomplish objectives with 3 to 6 months of fairly intensive treatment. Reducing the duration of adjuvant cyclophosphamide, methotrexate, fluorouracil (CMF) from 12 months to 6 months did not appear to have an adverse effect on long-term results. Factors such as dose intensity and early use of effective noncross-resistant agents may be much more important than the chronic administration of agents in suboptimal dosage. The narrower question as to whether advancing the time forward of adjuvant chemotherapy will make additional significant impact on survival cannot be answered yet but clearly is an important issue. There are several theoretical reasons why neoadjuvant treatment might be of particular benefit, and even if it ultimately transpires that breast cancer is not an ideal model disease for this approach, it does not preclude this particular technique for being effective in other types of malignancy.

Impact of dose-intense chemotherapy on the development of permanent drug resistance.

Using a somatic mutation theory for drug resistance, dose intense regimens are shown to be superior in increasing the likelihood of no doubly resistant cells and in curing the tumor. Consideration of this model suggests that dose intensity of early chemotherapy cycles should be calculated separately and correlated with treatment outcome.

Treatment of recurrent and metastatic head and neck cancer with cisplatin/etoposide/bleomycin.

Cisplatin/etoposide/bleomycin (DEB) was given as an outpatient regimen in a novel weekly schedule to 27 patients with recurrent and/or widely metastatic cancer of the head and neck region. Six of these patients also received mitomycin (DEB/M) when their disease failed to respond after at least three weekly DEB doses. All but three patients had been treated previously with radiotherapy directed to the primary site and regional nodal disease; four had also received chemotherapy with cisplatin or carboplatin. Before treatment with DEB, 19 patients had distant metastases. Of an intended 12 doses per patient, a mean of 8.2 was achieved. Myelosuppression was the major toxicity, with neutropenia in 45% of patients and significant anemia in 26%. The overall response rate to DEB in 27 patients was 59%, increasing to 70% after the addition of mitomycin. There were two complete and 17 partial responses. The median duration of response was 12 weeks and median survival was 6 months, with 20% of patients surviving 1 year. We conclude that the relatively short survival time together with the significant toxicity of the DEB/M regimen does not warrant its routine use in clinical practice. However, this regimen, or one patterned on it, should be evaluated in combination with radiotherapy as the initial treatment for selected patients with previously untreated head and neck cancer.

Further studies on substituted quinazolines and triazines as inhibitors of a methotrexate-insensitive murine dihydrofolate reductase.

Data are presented on the systematic analysis of thirty-five quinazoline and substituted triazine compounds as inhibitors of a methotrexate-insensitive form of dihydrofolate reductase purified from methotrexate-resistant L5178Y murine leukemia cells. Several of the compounds were found to be more potent inhibitors of this enzyme activity than was methotrexate. Two of the triazine compounds had IC50 values approaching 10nM, which is close to that of methotrexate for the normal drug-sensitive dihydrofolate reductase. In addition, some of these compounds, especially the triazines, exhibit a specificity of inhibition for the methotrexate-insensitive enzyme as compared to the normal methotrexate-sensitive dihydrofolate reductase derived from the same cell line. These compounds may, therefore, be potentially useful in the treatment of those methotrexate-resistant tumours which express an altered, methotrexate-insensitive dihydrofolate reductase.

Inhibition of a methotrexate-insensitive dihydrofolate reductase from L5178Y cells by substituted triazines and quinazolines.

Inhibition by a variety of substituted triazines and quinazolines of a methotrexate-insensitive form of dihydrofolate reductase from highly MTX-resistant L5178Y mouse leukemia cells was examined. Some of these compounds were significantly more potent than MTX (up to 100-fold). Two triazenes, terminally substituted with benzenesulfonylfluoride residues, were approximately 30-fold more potent than MTX. Quinazoline analogs of folic acid with alterations in different parts of the molecule varied in their potencies as inhibitors. Although none of the compounds tested was as potent as MTX against MTX-sensitive dihydrofolate reductases, these studies show that some types of folate antagonists have increased specificity against this MTX-insensitive dihydrofolate reductase. This finding increases the likelihood that it may be possible to produce compounds with marked specificity for the insensitive reductase. Such compounds might have utility in antifolate combinations designed to overcome methotrexate resistance.

Modelling the process of drug resistance.

Modelling the process of drug resistance can provide insight into such issues as the difference between intrinsic and acquired drug resistance. Other important biological questions such as whether resistance arises through selective or inducible events can also be addressed by reference to appropriate models. If drug resistant cells are produced by some type of inducible up regulation step then this has a number of qualifications for cancer chemotherapy. Selection theories of drug resistance imply that certain sequences of drugs will be superior to others something which can be tested by experiment.

Gas-chromatographic analysis of busulfan for therapeutic drug monitoring.

The development and validation of a gas chromatographic assay method for determination of total and free busulfan concentrations in human plasma for pharmacokinetic studies is reported. 1,6-Bis(methanesulfonyloxy)hexane, the internal standard, and a potential metabolite, 3-hydroxysulfolane, were synthesized. Plasma and plasma ultrafiltrate samples containing busulfan and internal standard were extracted with ethyl acetate and derivatized with 2,3,5,6-tetrafluorothiophenol prior to gas chromatographic determination. The 63Ni electron-capture detector provided a limit of detection of 0.0600 microgram/ml with a limit of quantitation of 0.100 microgram/ml busulfan in biological samples. Calibration curves were linear from 0.100 to 3.00 micrograms/ml in plasma (500 microliters) and 0.100 to 2.00 micrograms/ml in plasma ultrafiltrate (100 microliters). Extraction and derivatization yields ranged from 78.4% to 89.6% and 56.0% to 71.3%, respectively. Specificity of this assay for busulfan in the presence of its potential metabolites was demonstrated. Also, plasma samples containing co-administered drugs gave no response under these conditions. Clinical samples obtained following administration of a 1 mg/kg oral busulfan dose demonstrate the applicability of this method to analysis of total and free plasma concentrations.

The use of conventional salvage chemotherapy before dose-intensive cytotoxic therapy and autologous transplantation for aggressive-histology lymphoma: a case for re-evaluation.

The use of CSCT to judge suitability for DIT and AHSCT in patients with aggressive-histology lymphoma who recur after primary chemotherapy is a widespread practice that excludes many NHL patients from this potentially curative therapy. Surprisingly, little direct evidence exists to suggest that CSCT used in this way is a useful strategy. On the other hand, it is clear that many of these patients undergoing DIT and AHSCT will not be cured using any currently available strategy or technique, and a method to identify such patients would be most helpful. CSCT may or may not be the best way to do so. This is an important question, but currently there are insufficient data to give us a definitive answer. Clinical trials are needed to resolve the issue. If the utility of CSCT is not validated, it should be abandoned. If it is validated, however, we may begin to address ways in which CSCT may be given more effectively.

New information on drug resistance: implications for the adjuvant treatment of breast cancer.

We would suggest on the basis of our analysis that drug resistance still appears to represent a plausible explanation for drug treatment failure in adjuvant breast chemotherapy. It may not be the only factor, but, if present, clearly has to be circumvented if treatment results are to be improved. Since it seems most unlikely that a new wonder drug for breast cancer will emerge in the next few years, then it is to our existing armamentarium of antineoplastic agents that we will have to turn for improved therapeutic results. Fundamental questions will need to be asked about what indeed are the most appropriate agents to be used in combination chemotherapy protocols for this disease and what are the optimal dose ratios. Our own institutional experience in a number of areas has suggested that many chemotherapeutic protocols that are widely used represent significant underdosing and that achieving optimal results requires pushing therapeutic agents closer to the reasonable limits of tolerance. Enhanced techniques for patient support during programs of more intensive chemotherapy are now available, and it has also been our experience that patients tolerate briefer, intensive programs of chemotherapy better than they do protracted, less intensive protocols. The role of new drug combinations that incorporate synergistic or significant biochemical modulation effects (i.e., platinum-etoposide, 5-fluorouracil-leucovorin) need to be examined in the context of the management of breast cancer. We appear to have reached something of a plateau with existing protocols and approaches, and it is time to move ahead.

Variation in response to CCNU of glioblastoma multiforme in brain and cervical lymph node. Case report.

The case is reported of a patient in whom a cervical lymph node metastasis decreased in size while the primary intracranial glioblastoma continued to grow during chemotherapy with CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea). This is the first such case reported in humans. Possible explanations for this phenomenon are discussed.

Toxicity studies in thymidine kinase-deficient herpes simplex virus therapy for malignant astrocytoma.

Previous studies have shown that genetically engineered thymidine kinase (tk)-defective herpes simplex virus type 1 (HSV-1) can effectively and selectively destroy gliomas in animal models. The consequences of viral infection and tumor regression must be characterized before this therapy can be applied in human trials. To study the potential for long-term toxicity, immunocompetent rats harboring 9L gliosarcomas were injected intratumorally with a tk-defective HSV-1, KOS-SB, at titers that previously have been demonstrated to cause tumor regression. In animals surviving 3 months or longer following viral treatment, there was no evidence of persistent infection or inflammation in peritumoral brain tissue or in remote systemic organs studied with routine histological and immunocytochemical analyses. Polymerase chain reaction using primers specific for HSV-1 detected HSV-1 DNA in peritumoral tissue only in animals sacrificed within 3 months of viral injection. There was no evidence of HSV-1 DNA in systemic tissues at any time after treatment. We conclude that stereotactic intratumoral injection of tk-deficient HSV can be attempted for the treatment of brain tumors without risk of systemic infection or significant toxicity to normal brain or remote proliferating tissues.