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Previous data suggest that lipophilic statins such as fluvastatin and N-bisphosphonates such as zoledronic acid, both inhibitors of the mevalonate metabolic pathway, have anti-cancer effects in vitro and in patients. We have examined the effect of fluvastatin alone and in combination with zoledronic acid in the ATP-based tumour chemosensitivity assay (ATP-TCA) for effects on breast and ovarian cancer tumour-derived cells. Both zoledronic acid and fluvastatin showed activity in the ATP-TCA against breast and ovarian cancer, though fluvastatin alone was less active, particularly against breast cancer. The combination of zoledronic acid and fluvastatin was more active than either single agent in the ATP-TCA with some synergy against breast and ovarian cancer tumour-derived cells. Sequential drug experiments showed that pre-treatment of ovarian tumour cells with fluvastatin resulted in decreased sensitivity to zoledronic acid. Addition of mevalonate pathway components with zoledronic acid with or without fluvastatin showed little effect, while mevalonate did reduced inhibition due to fluvastatin. These data suggest that the combination of zoledronic acid and fluvastatin may have activity against breast and ovarian cancer based on direct anti-cancer cell effects. A clinical trial to test this is in preparation.

There is debate regarding the direct effect of bisphosphonates against visceral metastases from solid tumors, despite their proven efficacy against the skeletal complications of metastasis. The aim of this study was to determine whether zoledronic acid showed direct activity against five ovarian cell lines and tumor-derived cells, and whether addition of zoledronic acid to cytotoxic agents increased their cytotoxicity. In this study we used a standardized ATP-based tumor chemosensitivity assay (ATP-TCA) to measure the activity of alendronate, clodronate and zoledronic acid in five ovarian carcinoma cell lines and human solid tumors (breast, lung, ovarian, unknown primary carcinoma, and cutaneous and uveal melanoma) (n=34). We also tested the combination of zoledronic acid with paclitaxel and cisplatin in tumor-derived cells. All five cell lines exhibited greater sensitivity to bisphosphonates than the tumor-derived cells and in all five the IC50 for zoledronic acid was less than 4 muM. In the tumor-derived cells, zoledronic acid showed concentration-dependent inhibition with a median IC50 for all tumors tested of 17 muM and evidence of apoptosis (caspase activation). Simultaneous addition of zoledronic acid to cisplatin or paclitaxel showed no major increase in cytotoxicity. We conclude that the activity of bisphosphonates was greater in cell lines than in tumor-derived cells. However, the pattern of activity of bisphosphonates was the same in cell lines and tumor derived cells. This study suggests a direct, or possibly an indirect, effect of zoledronic acid and other nitrogen-containing bisphosphonates against neoplastic cells, but simultaneous addition with cisplatin or paclitaxel does not substantially increase the activity of the cytotoxic agent.

During the last two decades, novel nonclonogenic methods for pretherapeutic chemosensitivity testing have been developed that are likely to overcome major technical limitations of older assays such as low evaluability rates, low degree of standardization and reproducibility, lack of technical robustness, and poor methodological efficacy. Among these, the microplate adenosine triphosphate (ATP)-based tumor chemosensitivity assay (ATP-TCA) has gained particular merits for ex vivo chemosensitivity testing of native nonhematological tumors including cancers of the breast, ovary, gastrointestinal tract, cervix and corpus uteri, and lung; malignant melanomas; gliomas; sarcomas; and mesotheliomas. For this indication, the ATP-TCA can now be considered the best documented and validated technology. This assay, which is now commercially available, provides a highly reproducible, easy-to-handle kit technique; low technical failure rates; and a high methodological efficacy requiring only 1 x 106 tumor cells to test four to six different drugs or combinations. In ovarian and breast carcinomas, the predictive accuracy is > 90%, with a positive predictive value of 85-90% and a negative predictive value near 100%, respectively. In primary ovarian cancers, the ATP-TCA has been found to accurately predict both clinical response and survival. In two prospective clinical trials in patients with heavily pretreated ovarian cancer, chemotherapy individually selected by the ATP-TCA has been found to triple the response rates and nearly double the survival compared to empirically chosen regimens. Consequently, this assay, which is now under phase III evaluation, has successfully been used in new agent development to screen for novel chemotherapy regimens for the treatment of patients with breast and ovarian carcinoma and melanoma, respectively. This chapter highlights the recent preclinical and clinical experience with this promising technology and gives a detailed description of all the technical aspects of the ATP-TCA.

Trophoblast injury may be one of the possible causes of fetal distress associated with chemotherapy administered during pregnancy. The purpose of this study was to investigate the ex vivo chemosensitivity of normal trophoblasts (NTB) against commonly used antineoplastic agents. Using the newly developed ex vivo ATP-based trophoblast assay (ATP-TBA), 31 NTB freshly sampled from human placentas (gestational week 7-42) were tested against dactinomycin (Act-D), 5-fluorouracil (5-FU), 4-OOH-cyclophosphamide (4-HC), vincristine (VCR) and methotrexate (MTX) alone or in combination with calcium folate (LV). All agents were studied at concentrations relevant to clinical dosages normally used for chemotherapy of solid neoplasms. Of 31 samples studied with the ATP-TBA, 20 (65%) were evaluable. VCR, Act-D and 4-HC were the most active drugs with 55, 45 and 45% of samples responding ex vivo. Antimetabolites were less active, producing ex vivo response rates of 25 (MTX) and 20% (5-FU), respectively. MTX activity was largely neutralized by adding LV. The chemosensitivity of NTB showed considerable inter-individual variations and did not decrease with increasing gestational age. We therefore conclude that NTB of any gestational age exhibit considerable ex vivo sensitivity against common anticancer agents which is comparable to that observed for various solid tumors. The ATP-TBA may be helpful in planning future trials with both single agents and drug combinations in order to standardize and optimize chemotherapy during pregnancy., (Copyright 2002 Lippincott Williams & Wilkins.)

Liposomal doxorubicin (Caelyx/Doxil) has been shown to be active in around 20% of recurrent ovarian cancers. As yet, there is little clinical data on combinations of existing agents with liposomal doxorubicin, despite considerable clinical experience with soluble doxorubicin in combination. In this study, we have used an ATP-based tumor chemosensitivity assay to determine the relative efficacy of high concentrations of doxorubicin tested in combination with cisplatin, treosulfan, 5-fluorouracil (5-FU) or vinorelbine against cells obtained from recurrent ovarian tumor tissue. The results show little enhancement of the efficacy of high concentrations of doxorubicin by 5-FU, cisplatin, or treosulfan. However, vinorelbine+liposomal doxorubicin showed additive inhibition, and this combination is worthy of further testing in clinical trials.

We report on a 72-year-old patient developing Stewart-Treves syndrome (STS) of the right arm 9 years after curative irradiation for ipsilateral stage III breast cancer. Facing the poor track record of both irradiation and chemotherapy in this highly malignant lymphangiosarcoma, amputation was recommended but refused by the patient. Therefore, limb conserving-therapy using three courses of intra-arterial mitoxantrone (MX) and paclitaxel (PTX) was attempted. This novel chemotherapy protocol was selected by pretherapeutic ex vivo ATP-based chemosensitivity testing of autologous tumor tissue. The patient experienced complete response, which was subsequent histologically confirmed by compartment resection. When developing recurrent STS outside of the perfused area 6 months after primary therapy, the patient was retested and reinduced with three other courses of intraarterial MX/PTX which again produced durable complete remission. This case demonstrates the benefit of indivdualized therapy in this prognostically desperate disease allowing both limb conservation and maintained quality of life.

Chemotherapy of cancer based on cytotoxic agents has proved successful in the treatment of many cancers. The number of agents available to the oncologist has grown steadily and drug combinations are in widespread use. The perceived success of these combinations makes the introduction of new agents difficult. For any new agent, multiple phase II and III trials are likely to be needed. Since phase II/III trials usually only address single issues, the cost of introducing a new agent is substantial. Multiple studies are required with different tumor types to define the activity profile of a new drug, followed by adjusted combinations to define the role of the new drug in conjunction with older ones. Recent advances in the understanding of cancer at a molecular level are already leading to new agent design. The next problem is how to introduce and use these agents. One possible approach is to trial the drugs with tumor cells ex vivo, using a chemosensitivity assay such as the ATP-based chemosensitivity assay which is designed to mimic the situation within the tumor accurately enough to examine issues of dose response, sequence and timing in many different tumors. The avoidance of cell lines ensures relevance and the sensitivity of some of these methods allows large numbers of mechanistically logical permutations to be tested with material from small numbers of patients. The results may be used to choose the most effective combinations for clinical testing in a limited number of subsequent phase II/III trials, saving money and time, while permitting new agents to be introduced faster.

In the present study, our first experiences with the ATP Tumour Chemosensitivity Assay (ATP-TCA), a novel method for pretherapeutic drug testing, are reported. During one year, a total of 111 samples obtained from patients suffering from different gynaecological malignancies (ovary: 53, breast: 41, others: 17) were sent for chemosensitivity testing. The quality of 104 single cell suspensions was sufficient for further processing. Evaluability rates ranged from 71% (breast cancers and non-ovarian tumours) to 83% (ovarian cancers) with an average of 77%. Evaluability of recurrences was slightly better compared to primaries. A total of 18 different antineoplastic agents and 67 drug combinations was used for in vitro testing with an average of 12.4 drugs/combinations tested per each sample (ovary: 14.8, breast: 9.7, others: 10.2). In 70 of 111 cases, the ATP-TCA was followed by chemotherapy with 44 cases treated in respect of the assay results and 29 cases receiving an antineoplastic regime, which differed from the standard treatment protocol. Compared to untreated patients, recurrences were treated more frequently based on the ATP-TCA, with a majority receiving an alternative protocol as proposed by the assay. In ovarian carcinoma, chemosensitivity testing was of particular importance for the further therapy. 41 of 53 patients were treated by chemotherapy with 29 therapies basing on the results of the assay (alternative protocol: 17). The good correlation of the ATP-TCA and the individual clinical course of the patients was demonstrated by two case reports of recurrent ovarian carcinomas, one of them responding well to platinum-based combinations and the other presenting in vitro and in vivo platinum-resistance. In conclusion, our first experiences with the ATP-TCA were promising. Our results, however, warrant confirmation by studies with a sufficient number of cases and an extended observation period.

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.

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.