Systemic toxic effects associated with high-dose verapamil infusion and chemotherapy administration.

Aside from its more conventional uses as a cardiovascular drug, the calcium channel blocker verapamil has recently been added to chemotherapeutic regimens to reduce drug resistance in B-cell and other neoplasms that express the P-glycoprotein. We recently treated patients with continuous-infusion verapamil (0.15 mg/kg per hour to 0.60 mg/kg per hour) over a 5-day period in combination with continuous-infusion vincristine and doxorubicin plus oral dexamethasone. Seventy-one courses involving 35 hospitalized patients were prospectively studied for cardiovascular and other side effects. Cardiovascular side effects were observed most frequently and consisted of first-degree heart block, hypotension, sinus bradycardia, and junctional rhythms. We observed higher degree heart block, but the QRS interval remained narrow and the ventricular escape rate remained relatively normal. Effects on mean arterial pressure, heart rate, and PR interval were both time and dose related. Severe, symptomatic congestive heart failure was rarely observed. The most common noncardiovascular side effects were constipation, peripheral edema, and weight gain. All systemic toxic effects observed were easily treated or disappeared with either temporary or permanent discontinuation of the verapamil infusion or by a decrease in the dose of verapamil. We conclude that the cardiovascular side effects associated with continuous, high-dose intravenous verapamil therapy are significant and dose limiting but are rapidly reversible. Less cardiotoxic chemosensitizers are needed to reverse multidrug resistance in cancer.

In vitro evaluation of anticancer drugs against ovarian cancer at concentrations achievable by intraperitoneal administration.

High-dose intraperitoneal chemotherapy is a current developmental approach in the treatment of advanced ovarian cancer. Considerable pharmacologic data have been obtained on the intraperitoneal X time product for a number of agents administered by this route. We used the human tumor cloning assay (HTCA) to compare the activities of both standard and experimental agents used for intraperitoneal treatment. In vitro dose-survival curves were constructed for each drug over a two-log range of concentrations using fresh ovarian cancers from more than 50 patients. The mean concentration X time product (CXT) achievable in the intraperitoneal space after high-dose intraperitoneal drug administration was divided by the corresponding ID50 value (concentration of drug in vitro associated with 50% survival of tumor colonies) for each agent to calculate in vivo CXT: in vitro ID50 ratios. Using this approach, the standard agents, melphalan, cisplatin, and 5-FU were predicted to have similar efficacies by intraperitoneal administration, but doxorubicin and mitomycin were significantly inferior. Of the drugs tested, the new agent mitoxantrone was associated with the most favorable CXT to ID50 ratio and is, therefore, predicted to be particularly promising for intraperitoneal administration.

Application of a human tumor colony-forming assay to new drug screening.

The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and between laboratories, and evidence of ability to identify active compounds which were negative in the in vivo prescreen suggest that the human tumor colony-forming assay may be a valuable tool for antitumor drug screening. However, because of technical limitations inherent in the current assay methodology, this must be confined to selected tumor types and limited to screening on a moderate scale.

Development and applications of a human tumor colony assay for chemosensitivity testing.

Using the human tumor colony assay, the growth and chemosensitivity of clonogenic tumor cells present in fresh biopsies of human tumors can be investigated. Excellent evidence has been obtained that colonies grown in HTCA are comprised of tumor cells, and that clonogenic cells within tumor colonies have self-renewal properties (the defining feature of tumor stem cells). Clinical correlations have been made between in vitro chemosensitivity and the response of patients with metastatic cancer to chemotherapy. In a series of trials, HTCA has had a 71% true positive rate and a 91% true negative rate for predicting drug sensitivity and resistance respectively of cancer patients to specific chemotherapeutic agents. HTCA has also had several areas of application to new drug development and screening. Ongoing developmental research is needed to improve growth rates for many tumor types and to further improve assay methodology and thereby enhance its applicability to predictive drug sensitivity testing.

Human tumor colony assay and chemosensitivity testing.

The human tumor colony assay (HTCA; clonogenic or tumor stem cell assay) is an in vitro culture system employing semisolid medium support. Using HTCA, the growth and chemosensitivity of clonogenic tumor cells present in fresh biopsy specimens of human tumors can be investigated. Since this technique was described, there has been a marked increase in the direct study of human tumors in vitro. Excellent evidence has been obtained which establishes that colonies grown in HTCA are comprised of tumor cells and that clonogenic cells within tumor colonies have the property of self-renewal (the defining property of a tumor stem cell). Chemosensitivity testing with specific agents in HTCA has documented striking degrees of heterogeneity in drug sensitivity from patient to patient, even for tumors of the same histopathology. Clinical correlations have been made between in vitro chemosensitivity and the response of patients with metastatic cancer to chemotherapy. In a series of trials, HTCA has had a 71% true-positive rate and a 91% true-negative rate for predicting the drug sensitivity and resistance, respectively, of cancer patients to specific chemotherapeutic agents. The assay thus appears to be a prognostic factor which identifies chemosensitive patients and which in the future may allow some individualization of chemotherapy. HTCA has also had several areas of application to new drug development and screening. While results obtained have been encouraging, ongoing developmental research is needed to improve growth rates for many tumor types and to further improve, standardize, and simplify assay methodology. With such improvements, it appears likely that there will be a marked increase in the use of such in vitro human tumor assays for chemosensitivity testing and drug development.

Human myeloma in vitro colony growth: interrelationships between drug sensitivity, cell kinetics, and patient survival duration.

Ninety-seven patients with multiple myeloma evaluated serially had both a tritiated thymidine labeling index of bone marrow plasma cells (LI%) and in vitro myeloma stem cell culture performed. Thirty-three patients with myeloma colony growth had in vitro drug sensitivity testing carried out, 18 having in addition in vitro thymidine suicide determinations. The LI% and the likelihood of in vitro myeloma colony growth were highly correlated: the higher the LI%, the more likely was colony or cluster growth (p less than 0.001). The tritiated thymidine suicide of myeloma stem cells was usually very high. There was excellent correlation between in vitro and in vivo drug sensitivity. Both pretreatment drug resistance and selective sensitivity (e.g., interferon, bisantrene, methotrexate, vinblastine) at the time of relapse were accurately detected and correlated well with survival duration (p = 0.01 Wilcoxan). Although LI% and in vitro sensitivity were clearly independent variables, a high LI% (greater than 3%) plus in vitro resistance were associated with a subsequent survival duration of less than 6 mo. The studies allowed dissection of the complex interrelationship between cell kinetics and drug sensitivity.

Antitumor activity of didemnin B in the human tumor stem cell assay.

In vitro studies of the schedule dependency and cytotoxicity of didemnin B, a novel depsipeptide isolated from a Carribean tunicate (Didemnidae), were carried out in fresh tumor cells obtained from biopsies from 39 cancer patients using the human tumor stem cell assay. Two schedules of drug exposure were examined (1 h and continuous exposure in the agar). Tumor cells from nine of 26 patients (34.6%) showed reduced survival of tumor-colony forming units to 30% of control or less at the 0.01 microgram level in the continuous exposure studies. Cells from eight of 17 patients (47%) showed a similar degree of sensitivity to didemnin after a 1-h exposure to 0.1 microgram prior to plating. The median ID50 values were 4.2 X 10(-3) micrograms/ml and 46 X 10(-3) micrograms/ml for continuous and 1-h exposures, respectively. A clear dose-response relationship was observed with both dosage schedules. Comparison of the slopes of the continuous and 1-h exposures and of the ID50 of the drug schedules suggests that didemnin is a cell-cycle-non-specific cytotoxic agent. Significant in vitro antitumor activity was observed at low concentrations against carcinomas of the breast, ovary, and kidney, and also mesothelioma and sarcoma. These results can provide pharmacologic goals to be achieved in phase I clinical trial. Further in vitro testing should help select tumor types for study in phase II trials of this very promising new anticancer drug.

Studies of clonogenic human tumor stem cells.

In vitro tissue culture methods for cloning human tumor cells from fresh biopsy samples appear to provide a simple yet quantitative approach to study the biology and chemosensitivity of neoplastic cells which are likely to be responsible for micrometastasis and tumor progression in vivo. Use of this assay technique may provide a useful empirical approach to improve adjuvant therapy of cancer.

Salvage treatment of patients relapsing after breast cancer adjuvant chemotherapy.

We analyzed the efficacy of salvage therapy with systemic agents in 33 women who initially received adjuvant chemotherapy with Adriamycin and cyclophosphamide (AC) for early breast cancer. Relapses occurred at a median of 9 months after completion of adjuvant therapy, and six patients relapsed while receiving adjuvant treatment. Nonetheless, salvage treatment produced objective responses in three of ten patients (30%) receiving hormonal therapy alone, in five of 11 (45%) receiving AC plus hormonal therapy, and in none of 12 receiving cyclophosphamide, methotrexate, and 5-fluorouracil (P < 0.05). Survival from the time of relapse from adjuvant chemotherapy was superior for patients receiving hormonal therapy with or without AC chemotherapy compared to that of patients receiving cyclophosphamide, methotrexate, and 5-fluorouracil (P < 0.05), and the median survival time of responders is > 12 months. On the basis of these findings, we believe that patients relapsing after adjuvant chemotherapy should receive aggressive treatment employing Adriamycin combination chemotherapy along with hormonal therapy.

Cell kinetic analysis of human tumor stem cells.

The application of the tumor stem cell assay to the study of human tumor cell kinetics has the potential for major advances in our knowledge of proliferative characteristics of clonogenic human tumor cells. From simultaneous evaluation of in vitro drug sensitivity, in vitro doubling time, and the thymidine suicide index, plus flow cytometry, cytogenetic analysis, and assessment of differentiation markers, substantial insights into the basic biology of tumor cell growth may be attained. As discussed in other chapters, using modifications of the two-layer system, one can assess both local cell-mediated and humoral factors that might influence in vitro kinetics and drug sensitivity. The next few years should see the acquisition and integration of valuable new information that should allow more rational approaches to the treatment of tumors by taking full advantage of information on both kinetics and drug sensitivity of clonogenic human tumor cells.

Applications of the human tumor stem cell assay to new drug evaluation and screening.

The potential applications of the human tumor stem cell assay to primary drug screening and Phase I-II new drug evaluation are of very broad scope and have not been completely addressed in this exploratory analysis. As in most other areas of science, careful and stepwise testing in many laboratories will be required to confirm or refute the utility of these approaches. Of the two areas discussed, the applications to new drug screening are still most uncertain and subject to change. From my perspective, in vitro growth conditions, for clonogenic human tumor cells, are perhaps at a similar stage of development as culture techniques for bacterial cells were in the 1930s. Availability of those techniques, and a combination of good ideas, serendipidy, and hard work led to the subsequent development of penicillin and other antibiotics which now can be used to cure most forms of infectious disease. The value of a simple and reproducible assay for screening therefore cannot be overemphasized. Applications of the tumor stem cell assay to Phase I-II drug evaluation already show preliminary signs of utility. Given this background and the more limited preclinical data on other agents now under study, broadened testing of this approach to new drug screening appears warranted. If use of an in vitro approach such as this proves successful for both primary and secondary screening, it would not be surprising if significant qualitative and quantitative changes occurred in the areas of new drug development and cancer clinical trials.