Heterogeneity of chemosensitivity of metastatic cutaneous melanoma.

Advanced melanoma has a poor prognosis and chemotherapy provides little benefit for most patients. This may be related to heterogeneity of chemosensitivity as well as frequent constitutive resistance to individual cytotoxic drugs. We have therefore examined the heterogeneity of chemosensitivity in metastatic cutaneous melanoma specimens using an ex vivo ATP-based chemosensitivity assay (ATP-TCA). Melanoma deposits (n=55) in skin or lymph node were tested using the ATP-TCA, performed in three separate laboratories. Analysis of the data collected (based on an arbitrary sensitivity index < 300) shows considerable heterogeneity of chemosensitivity. The most active single cytotoxic agents in the assay were identified as cisplatin, treosulfan, paclitaxel, vinblastine, gemcitabine and mitoxantrone. There was also a limited direct inhibition of melanoma cell growth by interferon-alpha2b, although this agent is known to have a number of indirect biological antitumor effects. Exposure of tumor cells to combinations of drugs at the concentrations tested as single agents showed the most active combinations to be treosulfan+gemcitabine, cisplatin+paclitaxel and vinblastine+paclitaxel. There was considerable heterogeneity of chemosensitivity: some tumors responded well to one agent or combination, while others showed no response to this and instead responded to one of the alternatives tested. Occasional highly resistant tumors showed no response to any of the single agents or combinations tested. The degree of heterogeneity observed suggests that the ATP-TCA could be used to select patients who might benefit from specific chemotherapeutic agents alone or in combination. This provides the rationale for future randomized controlled trials of ATP-TCA-directed chemotherapy versus physician's choice to determine whether assay-directed chemotherapy can improve patient response and survival.

Application of a new high sensitivity luminometer for industrial microbiology and molecular biology.

A compact new luminometer (FB12) has been developed based on a 370-630 nm photon counter and measuring chamber that can accommodate a range of sample formats. The FB12 permits measurements as low as 1000 molecules of luciferase in reporter gene assays. Its sensitivity for ATP is limited by reagent background. If ATP assay reagents had no chemical background, 2 fg of ATP could be detected using 3 SD of instrument background as the detection limit. The FB12 has a dynamic range of six decades and operates under its own microprocessor programme or protocol-based PC software that is integrated with Microsoft(R) Excel(R). An injector port above the sample measuring position allows connection of external reagent injectors. Applications are performed using protocols provided with the FB12 or user defined protocols. Examples are presented that illustrate use of the instrument for research and industrial applications.

Use of an ex vivo ATP luminescence assay to direct chemotherapy for recurrent ovarian cancer.

Chemotherapy for recurrent ovarian carcinoma (ROC) produces response rates of 10-80% depending on the prevalence of platinum resistance. Most patients relapse within 1 year and median progression-free survival (PFS) is generally no more than 6 months. Previous pretherapeutic chemosensitivity assays mostly failed to improve the outcome of patients with ROC. Newly developed ATP assays show promising retrospective correlation with clinical outcome. We report here the first results of ATP assay-directed chemotherapy in patients with ROC. Therapy was selected by the ATP tumor chemosensitivity assay (ATP-TCA) in a prospective open-label pilot trial for ROC. Objective response rate (ORR), PFS and overall survival (OAS) of the first 25 evaluable patients were retrospectively compared with those of 30 others having similar characteristics who were treated empirically within the same period. The actuarial median observation times were 80 weeks for the ATP-TCA group and 83.5 weeks for the control group, respectively. In the control group, a 37% ORR [two complete responses (CR) and nine partial responses (PR)] was followed by a median PFS of 20 weeks and a median OAS of 69 weeks, mainly related to the use of single-agent chemotherapy. The ORR in the ATP-TCA group was 64% (eight CR and eight PR) (p=0.04) with the majority of responses (11 of 16) achieved with novel combinations. The median PFS in this group was 50 weeks (p=0.003) and the median OAS was 97 weeks (p=0.145). Survival of responding patients was similar in both groups. Chemotherapy guided by the ATP-TCA produced a greater benefit with regard to both ORR and PFS in platinum-refractory patients. ATP-TCA-directed chemotherapy for ROC compares favorably with chemotherapy chosen by a clinician and often leads to the choice of novel drug combinations. These promising results now warrant confirmation by prospective randomized trials.

Measurement of cytotoxicity by ATP-based luminescence assay in primary cell cultures and cell lines.

Drug discovery and toxicological safety testing share a need for dependable in vitro cellular toxicity tests. Ideally such tests should be objective, quantitative, reproducible and able to lend themselves to automation. A number of assays fulfil these criteria well, but recently it has become clear that the molecular phenotype of the cell tested and the complex interplay between different cell types can radically alter the response to individual agents. The differences observed between primary cell cultures and cell lines make it preferable to use primary cultures for assessment of toxicity, yet the problems of using primary cell cultures are considerable as the number of cells available for testing is often small. Recently, we have developed a short-term cell culture assay based on the detection of ATP by the luciferin-luciferase reaction. Four drugs/agents can be tested in triplicate at seven dilutions in one 96-well microplate with 1000 cells/well in the case of cell lines, or 10,000 cells/well for primary tumour tissue. The small number of cells required is a major advantage of this method. Initially developed as a tumour chemosensitivity assay, the assay has shown considerable promise as a general in vitro toxicity assay allowing both cell lines and primary tissue cultures to be tested. Heterogeneity of sensitivity is present in benign tissue biopsies as well as tumours. Molecular alterations within the cell and the interplay of different cell types have been addressed in a number of different model systems using the assay, suggesting that this technology may have more general application.

Mitoxantrone combined with paclitaxel as salvage therapy for platinum-refractory ovarian cancer: laboratory study and clinical pilot trial.

This report describes preclinical and early clinical investigations of the mitoxantrone/paclitaxel combination (NT) for patients with platinum-refractory ovarian cancer. The preclinical activity of NT was studied ex vivo, evaluating native tumor specimens with the ATP tumor chemosensitivity assay. Of 24 tumors tested, 20 (83%) were sensitive to NT, whereas 7 (29%) responded to mitoxantrone and 8 (33%) responded to paclitaxel. In the majority of tumors assayed (19 of 24), potentiating or major independent effects between both agents were found. Subsequently, a clinical pilot trial of NT was initiated for patients with platinum-refractory ovarian cancer. Patients had failed one to four (median, two) prior chemotherapy regimens. In 11 cases, NT was administered every three weeks with 8 mg/m2 mito-xantrone and 180 mg/m2 paclitaxel (NT-I). Seven patients were treated biweekly with 6 mg/m2 mitoxantrone and weekly with 100 mg/m2 paclitaxel (NT-II). During 92 NT courses, myelosuppression with leucopenia, anemia, and thrombocytopenia was the limiting toxicity, occurring more frequently with NT-II. No patient required hospitalization due to any life-threatening complication. Five complete and nine partial remissions were observed with both NT-I and NT-II, accounting for an overall 78% response rate, with a median progression-free survival of 40 weeks. One patient showed early progression during therapy. Currently, three patients (NT-I, two; NT-II, one) have died due to progressive relapsed ovarian cancer, so that the median overall survival is not reached after a median follow-up of 40.5+ weeks. Both schedules were found to be equal in terms of response rate and overall survival. NT is highly active and practical for salvage treatment of ovarian cancer. NT-II may be preferred due to both clinical activity and patients' acceptance. However, NT-I seems to be a less myelotoxic alternative. Both schedules warrant further clinical investigation.

Correlation of the clinical response to chemotherapy in breast cancer with ex vivo chemosensitivity.

Chemotherapy for breast cancer is given on the basis of empirical information from clinical trials, an approach which falls to take into account the known heterogeneity of chemosensitivity between patients. Previous attempts to determine chemosensitivity ex vivo have been disappointing, but in this study results from a newly developed tumor chemosensitivity assay (TCA) have been correlated prospectively with patient response. In this study, we have used heterogeneity data for standard regimens obtained from 116 breast TCAs to set sensitivity/resistance thresholds which were then used to interpret the results from those with known clinical responses. Assay evaluability was 97% in surgical biopsies. Clinical follow-up of stage III/ IV assessable disease was obtained from 27 breast tumors which were successfully tested for chemosensitivity, including 13 needle biopsies. The ATP-TCA assay predicted response correctly in 22 out of 29 (76%) tumors with clinically evaluable disease, suggesting that it is capable of predicting outcome in individual patients. Assays were performed in seven patients before and after chemotherapy using residual or recurrent tumor tissue. Four cases with initial sensitivity showed a decrease in sensitivity within 6 months of starting chemotherapy, while two others without clinical resistance were still sensitive by TCA. All nine courses of therapy given on the basis of TCA sensitivity resulted in partial or complete responses. Controlled trials of TCA-directed treatment against standardized empirical therapy should be conducted before this technology is widely adopted to assess its impact on rates of response, survival and the cost of treatment.

Ascorbic acid (vitamin C) improves the antineoplastic activity of doxorubicin, cisplatin, and paclitaxel in human breast carcinoma cells in vitro.

Utilizing a microplate ATP bioluminescence assay, two human breast carcinoma cell lines, MCF-7 and MDA-MB-231, were tested against doxorubicin (DOX), cisplatin (DDP), and paclitaxel (Tx) alone and in combination with ascorbic acid (Vit C). In both cell lines, Vit C exhibited cytotoxic activity at high concentrations (i.e. 10(2)-10(3) microM). Both cell lines also were resistant to DOX. MCF-7 was found to be DDP-resistant, MDA-MB-231 was moderately sensitive to DDP. Both cell lines were strongly sensitive to Tx. Vit C both at non-cytotoxic (1 microM) and moderately cytotoxic concentrations (10(2) microM) improved the cytotoxicity of DOX, DDP, and Tx significantly. Combination effects between Vit C and DDP or Tx were partly synergistic and partly additive or subadditive whereas a consistent synergism was found between Vit C and DOX. The mechanisms by which Vit C potentiates the cytostatics studied are yet unclear and should be evaluated further.

Heterogeneity of in vitro chemosensitivity in perioperative breast cancer cells to mitoxantrone versus doxorubicin evaluated by a microplate ATP bioluminescence assay.

Apart from clinical trials, mitoxantrone (MX) is rarely used in breast cancer (BC) due to the anticipated anthracycline cross-resistance. We have examined this drug versus doxorubicin (DOX) using data obtained from in vitro microplate ATP tumor chemosensitivity assays (ATP-TCA) of BC cells which were derived from 55 chemotherapy-naive patients at time of primary surgery. Both drugs were tested at 6 different concentrations ranging from 6.25% to 200% peak plasma concentration in vivo (PPC). Differences between DOX and MX observed for mean IC50, IC90, and a sensitivity index (SI) were not statistically significant. In vitro response rates were 44% for DOX and 52% for MX. 34 of 52 eligible assays (65%) showed comparable activity of both drugs whereas a lack of cross-resistance was observed in the remaining 18 (35%) tumors as indicated by differences for SI. Cumulative concentration-response plots of tumors responding in vitro with a > or = 50 percent or > or = 90 percent tumor cell inhibition showed a strong dose-dependence for both DOX and MX at concentrations which normally can be achieved within clinical tumors (i.e. 6.25%-50% PPC). At higher concentrations, however, cytotoxicity of DOX and MX could not be improved by further in vitro dose escalation. Moreover, a substantial proportion of BC specimens (DOX: 48.1%; MX: 40.4%) did not experience a > or = 90 tumor cell inhibition at 200% PPC. In conclusion, in vitro results obtained by ATP-TCA indicate that there is no cross-resistance between MX and DOX in a substantial proportion of BC patients. This may be clinically useful and suggests that combinations including MX should be tested in patients clinically resistant to DOX containing regimens. Since both drugs produced sigmoidal concentration-response curves, dose escalation beyond a certain point may not produce increased sensitivity.

Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian carcinoma.

An ATP luminescence assay (TCA 100) was used to measure chemotherapeutic drug sensitivity and resistance of dissociated tumor cells cultured for 6 days in serum-free medium and 96-well polypropylene microplates. Studies were performed with surgical, needle biopsy, pleural, or ascitic fluid specimens using 10,000-20,000 cells/well. ATP measurements were used to determine tumor growth inhibition. Single agent and drug combinations were evaluated using the area under the curve and 50% inhibitory concentration (IC50) results for a series of test drug concentrations. The ATP luminometry method had high sensitivity, linearity, and precision for measuring the activity of single agents and drug combinations. Assay reproducibility was high with intraassay and interassay coefficients of variation of 10-15% for percentage of tumor growth inhibition, 5-10% for area under curve, and 15-20% for IC50 results. Good correlation (r = 0.93) between the area under the curve, and IC50 results was observed. Cytological studies with 124 specimens demonstrated selective growth of malignant cells in the serum-free culture system. Studies with malignant and benign specimens also showed selective growth of malignant cells in the serum-free medium used for assay. The assay had a success rate of 87% based on criteria for specimen histopathology, magnitude of cell growth, and dose-response drug activity. Cisplatin results for ovarian carcinoma are presented for 81 specimens from 70 untreated patients and 33 specimens from 30 refractory patients. A model for interpretation of these results based on the correlation of clinical response with the area under the curve and IC50 results indicates that the assay has > 90% accuracy for cisplatin resistance of ovarian carcinoma. Additional studies are in progress to evaluate the clinical efficacy of this assay.

In vitro activity of titanocenedichloride versus cisplatin in four ovarian carcinoma cell lines evaluated by a microtiter plate ATP bioluminescence assay.

Titanocenedichloride (MKT 4) is a novel anticancer drug with a broad spectrum of activity in mammalian tumors. We investigated the anticancer efficacy of MKT 4 versus cisplatin and its chemomodulation by buthionine sulfoximine (BSO) in four different human ovarian carcinoma (OvCA) cell lines derived from both primary (A2780. OTN 14) and recurrent tumors (SKOV-3 and OV-MZ-1b) using an in vitro microplate ATP bioluminescence assay (ATP-TCA). Sensitivity against cisplatin was higher in A2780 and OTN 14 compared with MKT 4, whereas the opposite was found in SKOV-3 and OV-MZ-1b cells. In A2780, SKOV-3 and OV-MZ-1b, the cytotoxicity of both agents could be effectively improved by BSO with supraadditive effects observed for MKT 4 in all three cell lines. In OTN 14, however, BSO treatment failed to increase the cytotoxicity of both cisplatin and MKT 4. These results suggest antineoplastic activity of MKT 4 in cisplatin-sensitive and mainly in cisplatin-resistant OvCA cells which can be significantly modulated by BSO-mediated glutathione depletion. Since antineoplastic activity of both cisplatin and MKT-4 observed in OTN 14 could not be reversed by BSO, other mechanisms of drug resistance different from the glutathione redox cycle are likely to be important for both metal compounds.

Methotrexate chemosensitivity by ATP luminescence in human leukemia cell lines and in breast cancer primary cultures: comparison of the TCA-100 assay with a clonogenic assay.

Chemosensitivity assays are widely used to predict the ability of tumor cell lines to respond to potential or existing cytotoxic drugs. In this study we have compared the cell cloning assay first described by Salmon and Hamburger with a recently developed assay which measures viable cell number by ATP luminescence. Methotrexate (MTX) was chosen as the test agent, since cell lines with varying degrees of sensitivity to this agent were readily available. The results shown good correlation between the two assays, both of which are able to discriminate between the various cell lines used. MTX inhibition of primary breast carcinomas and cell lines shows a steep dose-response curve with a threshold concentration above which increasing dose does not increase sensitivity. In solid tumors, the plateau is usually reached at a level well below 100% inhibition. The ATP luminescence assay allows discrimination of MTX sensitivity between breast carcinomas and has considerable technical advantages over the cloning assay.

Taxol inhibits growth of mesothelioma xenografts.

BACKGROUND: In vitro tests measuring inhibition of adenosine triphosphate activity predicted three mesothelioma xenografts would be sensitive to taxol. PURPOSE: The in vivo therapeutic efficacy of taxol was tested in nude mice carrying the subcutaneous tumors. METHODS: Once tumor growth reached 100mm3 in size, intraperitoneal taxol, 30 mg/kg, on a day 1, 4, and 8 schedule, was administered to mice bearing subcutaneous mesothelioma xenografts. RESULTS: Taxol inhibits the growth of all three cell lines. It produces actual tumor regression including some complete responses. CONCLUSIONS: Taxol is an active drug against mesothelioma. The in vitro cell lines and the in vivo system are useful tools for screening and developing new treatments for mesothelioma.

TCA-100 tumour chemosensitivity assay: differences in sensitivity between cultured tumour cell lines and clinical studies.

The BATLE LE TCA-100 tumour chemosensitivity assay has been used to evaluate chemotherapeutic drug sensitivity of cultured tumour cell lines. Studies were performed using test drug concentrations calibrated to discriminate sensitivity and resistance of clinical specimens. Strong sensitivity which appeared to be inconsistent with clinical experience was detected for some drugs and cell lines. Findings of strong sensitivity were consistent with basic differences between sensitivity testing cultured cell lines and clinical specimens. Results with cell lines frequently may not apply directly to clinical applications. Characterization of differences between cell lines and clinical specimens may assist in application of cell line findings to clinical trials.

In vitro activity of titanocenedichloride versus cisplatin and doxorubicin in primary and recurrent epithelial ovarian cancer.

BACKGROUND: Titanocenedichloride (MKT 4) is a new antineoplastic metal complex with proven activity in several experimental tumors. MATERIAL AND METHODS: In the present study, the cytotoxic activity of titanocenedichloride in fourteen primary and twelve recurrent ovarian carcinomas (OvCA) was evaluated by an in vitro adenosine triphosphate (ATP) bioluminescence assay. RESULTS: In primary tumors, MKT 4 was found to be at least as effective as cisplatin (DDP) and doxorubicin (DOX). In samples derived from pretreated patients, titanocenedichloride was even more active. In both groups of tumors, a lack of cross resistance between the two metal compounds as well as between MKT 4 and DOX was apparent. The new agent was found to be active in eight of seventeen DDP-resistant (primaries: n = 4; recurrences n = 4) and also eight of seventeen DOX-resistant tumors (primaries: n = 4; recurrences n = 4). CONCLUSIONS: These results indicate a remarkable in vitro activity of titanocenedichloride in native OvCA specimens, even in those exhibiting resistance against cisplatin or doxorubicin. The putative role of this novel drug for the future therapy of OvCA should be evaluated by additional in vitro and in vivo studies.

In vitro activity of titanocenedichloride in human renal cell carcinoma compared to conventional antineoplastic agents.

In the present study, the in vitro activity of titanocenedichloride, a novel anticancer metal complex, in eleven primary renal cell carcinoma (RCC) specimens was evaluated by an adenosine triphosphate (ATP) bioluminescence assay. Compared to standard antineoplastic agents such as cisplatin, doxorubicin, mitoxantrone and vinblastine, titanocenedichloride was found to exhibit higher cytotoxicity. Four tumors showed strong sensitivity to the new agent. Three of them were resistant to conventional cytostatics. These findings indicated a lack of cross-resistance between titanocenedichloride and both natural compounds as well as platin analogues. Since mitoxantrone was also found to exhibit marked antineoplastic activity with no evidence of cross-resistance to titanocenedichloride, the combination of both drugs in RCC should be evaluated further with additional in vitro studies.

The influence of storage on cytotoxic drug activity in an ATP-based chemosensitivity assay.

The use of viability assays to assess the effect of antineoplastic agents on cell lines and tumor cells is an important investigative tool and may have clinical relevance. Such assays require very small quantities of drugs and it is the practice of many laboratories to freeze aliquots of drugs for use in these assays as required. We have investigated the stability of 11 different agents in an ATP-based chemosensitivity assay which is being evaluated for clinical use. The results show that most drugs maintain their biological activity well when frozen at -20 degrees C for periods up to 24 months, or occasionally at room temperature. However, 4-hydroperoxycyclophosphamide and mitomycin C are exceptions to this rule, and should not be kept frozen for more than 2-3 months. Cisplatin is unstable when frozen and then thawed, but maintained activity at room temperature for at least 6 months. Since biological activity may not correlate completely with chemical stability, further studies on the effect of storage are required, but it seems unlikely that the appropriate use of frozen aliquots is a major source of error in tumor chemosensitivity assays.

Expression of the p53 tumour suppressor gene product is a determinant of chemosensitivity.

Many cytotoxic agents act by causing DNA damage, and the p53 tumour suppressor gene is known to be involved in the cellular response to DNA damage. Since inactivation of p53 is common in many tumours, we wondered if this would affect the sensitivity of cancer cells to cytotoxic agents. We have shown that this is indeed the case in transformed mouse cell lines with and without a mutated p53 gene; p53 "knockout" mouse fibroblasts, and normal human skin fibroblasts treated with an anti-sense p53 oligonucleotide. In addition, we have demonstrated a correlation between p53 protein expression in human breast cancer specimens and their chemosensitivity. The results show that inactivation or mutation of p53 renders cells more sensitive to those cytotoxic drugs whose primary mechanism of action is DNA damage.

Heterogeneity of chemosensitivity in human breast carcinoma: use of an adenosine triphosphate (ATP) chemiluminescence assay.

Cancer chemotherapy is currently given to patients with breast carcinoma on the basis of data from response rates in patients with advanced disease, or from the results of clinical trials of adjuvant therapy. However, individual tumours may vary in their response to particular cytotoxic drugs: optimal therapy for a population of patients may not be the correct treatment choice in individual cases. In this study we have used an ATP-based non-clonogenic chemosensitivity assay (TCA-100) to investigate the heterogeneity of chemosensitivity of human breast carcinoma to a number of cytotoxic drugs, both as single agents and in combination. Tissue was obtained from 33 patients. Most samples were excision biopsies, but sufficient tumour cells were obtained from three needle biopsies and three pleural effusions for assays to be performed. The results show wide variation in the response of individual breast tumours to single agents, but most tumours show sensitivity to the commonly used combination regimens. The TCA-100 assay may provide useful information to support the choice of regimen for breast cancer chemotherapy.

Microbioluminometry assay: determination of erythromycin activity in plasma or serum.

A microbioluminometry assay (MBA) was developed for the quantitative analysis of erythromycin activity in human plasma or serum. The MBA method is adapted from turbidimetric methods and utilizes an enzyme-catalyzed bioluminescence reaction to quantitate the growth of Staphylococcus aureus in liquid culture medium. Statistical analysis of data obtained in method validation studies and in more than 500 assays of standard curve and control samples demonstrates consistent reproducibility and accuracy within theoretical limits. The MBA was shown to be more sensitive than agar diffusion assays with a lower limit of sensitivity less than 20.0 ng/mL and coefficients of variation less than 10%. Cumulative results of 178 assays of spiked control plasma samples in the range of 0.14-2.18 micrograms/mL show 11.2% of individual determinations are greater than +/- 15% the expected value, and 5.6% of individual determinations are greater than +/- 20% the expected value. Bioavailability profiles obtained with MBA are consistent with reported data for erythromycin. Values for 206 subject samples analyzed by MBA and agar diffusion assays showed a high degree of correlation (r = 0.9525) between the two methods. The MBA technique provided high sample throughput because of the use of microtiter plate technologies; it is also economical since it requires less sample and reagents.

Natural killing target antigens as inducers of interferon: studies with an immunoselected, natural killing-resistant human T lymphoblastoid cell line.

The human T lymphoblastoid cell line CEM was subjected to immunoselection by co-culture with peripheral blood mononuclear cells (PBMC) for resistance to natural killer (NK) cell-mediated lysis. The NK susceptibility of the resulting subline, CEM.NKR, was 8.4 to 20.6% of that of CEM when PBMC or adherent cell-depleted PBMC were used as effector cells, and -7.1 to 12.1% of that of CEM when Percoll gradient-enriched large granular lymphocytes (LGL) were used. However, CEM and CEM.NKR exhibited comparable sensitivity to antibody-dependent cellular cytotoxicity. Unlabeled CEM was eight- to 32-fold more effective than unlabeled CEM.NKR in inhibiting the NK lysis of labeled CEM target cells, and CEM bound 1.9 to 3.9-fold more Percoll gradient-enriched LGL than CEM.NKR in single cell-binding assays, suggesting that the NK-resistant variant has lost the expression of NK target antigens. However, CEM.NKR was comparable to CEM in its ability to induce interferon (IFN)-alpha production by PBMC in vitro, and the NK-resistant variant maintained its susceptibility to the antiproliferative effects of IFN-alpha, indicating that these phenomena may be mediated by molecules other than NK target structures. Comparison of CEM and CEM.NKR by indirect immunofluorescence with monoclonal antibodies specific for leukocyte antigens and the transferrin receptor, and by microcytotoxicity typing for HLA-A and B specificities, revealed no major differences.