The effect of tricyclic antidepressants on cutaneous melanoma cell lines and primary cell cultures.

The tricyclic antidepressants have previously been shown to exert activity against glioma cells in vitro. Initial studies in cell lines suggested that this might extend to melanoma cells. We have therefore conducted a study in primary cell cultures from metastatic cutaneous melanoma deposits using a well established ATP-based tumour chemosensitivity assay to confirm and extend these findings. Two cell lines and eight primary cell cultures from metastatic melanoma deposits were exposed to three tricyclic drugs, amitriptyline, nortriptyline and clomipramine, at concentrations ranging from 200 to 6.25 µmol/l in the ATP-based tumour chemosensitivity assay. All three drugs showed activity, although nortriptyline was more active than clomipramine or amitriptyline in both cell lines and primary cell cultures, with an IC50 of 9, 27 and 33 µmol/l, respectively. Tricyclic agents show activity against melanoma in vitro. This could be related to the lysosomal effects based on their cationic amphiphilic properties, or effects at the mitochondrial membrane.

Quality assurance and good laboratory practice.

Poor cell culture practice leads to poor science due largely to issues of cross-contamination between cell lines and of microbial contamination, but can be avoided by careful quality control and good laboratory practice. This chapter provides a brief and practical outline of the steps needed to mitigate the risks associated with poor cell culture practice. Good Laboratory Practice (GLP) is a set of principles that provides a framework within which laboratory studies are planned, performed, monitored, recorded, reported, and archived to ensure the reliability of data generated within a compliant laboratory. A key feature of this is the generation of quality-control methods and data management within the cell culture laboratory.

The molecular basis of the chemosensitivity of metastatic cutaneous melanoma to chemotherapy.

BACKGROUND: Chemotherapy benefits relatively few patients with cutaneous melanoma. The assessment of tumour chemosensitivity by the ATP-based tumour chemosensitivity assay (ATP-TCA) has shown strong correlation with outcome in cutaneous melanoma, but requires fresh tissue and dedicated laboratory facilities. AIM: To examine whether the results of the ATP-TCA correlate with the expression of genes known to be involved in resistance to chemotherapy, based on the hypothesis that the molecular basis of chemosensitivity lies within known drug resistance mechanisms. METHOD: The chemosensitivity of 47 cutaneous melanomas was assessed using the ATP-TCA and correlated with quantitative expression of 93 resistance genes measured by quantitative reverse transcriptase PCR (qRT-PCR) in a Taqman Array after extraction of total RNA from formalin-fixed paraffin-embedded tissue. RESULTS: Drugs susceptible to particular resistance mechanisms showed good correlation with genes linked to these mechanisms using signatures of up to 17 genes. Comparison of these signatures for DTIC, treosulfan and cisplatin showed several genes in common. HSP70, at least one human epidermal growth factor receptor, genes involved in apoptosis (IAP2, PTEN) and DNA repair (ERCC1, XPA, XRCC1, XRCC6) were present for these agents, as well as genes involved in the regulation of proliferation (Ki67, p21, p27). The combinations tested included genes represented in the single agent signatures. CONCLUSIONS: These data suggest that melanoma chemosensitivity is influenced by known resistance mechanisms, including susceptibility to apoptosis. Use of a candidate gene approach may increase understanding of the mechanisms underlying chemosensitivity to drugs active against melanoma and provide signatures with predictive value.

The effect of pentamidine on melanoma ex vivo.

Pentamidine is a small molecule inhibitor of the Ca(+)-binding protein S100B and disrupts the S100B-p53 protein-protein interaction; this is thought to restore wild-type p53 tumour suppressor function in melanoma. Additional anticancer effects may be the result of inhibition of regenerating liver family phosphatases. In this study, we have used a standardized ATP-tumour chemosensitivity assay to investigate the effect of pentamidine on cells derived from 18 skin melanoma samples and one uveal melanoma sample. The cells were tested at six concentrations from which the IC(50) and IC(90) were calculated. To allow comparison between samples, an index(sum) was calculated based on the percentage of tumour growth inhibition at each concentration. Of the skin melanoma samples tested, 78% exhibited an index(sum) less than 300 indicating strong inhibition. The median index(sum) of 237 also indicates considerable activity against these samples. The median IC(90) (30.2 micromol/l) may be clinically achievable in a proportion of patients. The uveal melanoma sample exhibited an index(sum) of 333 indicating moderate inhibition, and 86% inhibition at test drug concentration (37.96 micromol/l). These results show that pentamidine has activity against melanoma, and support the prospect of its development for therapeutic use.

Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC).

BACKGROUND: NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors. METHODS: The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA) and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array following extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: There was considerable heterogeneity between tumors within the ATP-TCA, and while this showed no direct correlation with individual gene expression, there was strong correlation of multi-gene signatures for many of the single agents and combinations tested. For instance, docetaxel activity showed some dependence on the expression of drug pumps, while cisplatin activity showed some dependence on DNA repair enzyme expression. Activity of both drugs was influenced more strongly still by the expression of anti- and pro-apoptotic genes by the tumor for both docetaxel and cisplatin. The doublet combinations of cisplatin with gemcitabine and cisplatin with docetaxel showed gene expression signatures incorporating resistance mechanisms for both agents. CONCLUSION: Genes predicted to be involved in known mechanisms drug sensitivity and resistance correlate well with in vitro chemosensitivity and may allow the definition of predictive signatures to guide individualized chemotherapy in lung cancer.

Activity of mevalonate pathway inhibitors against breast and ovarian cancers in the ATP-based tumour chemosensitivity assay.

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.

Effect of culture conditions on the chemosensitivity of ovarian cancer cell lines.

The aim of this study was to define the chemosensitivity profile of a series of human ovarian cancer cell lines representing the human primary ovarian tumours under altered culture conditions and to compare the results with those from tumour-derived cells. In this study, we used a standardized ATP-based tumour chemosensitivity assay to measure the activity of cytotoxics in the seven ovarian carcinoma cell lines and ovarian tumour-derived cells. The use of adherence-free polypropylene plates and a serum-free medium slowed down cell proliferation in all cell lines tested, mimicking the slow growth rate of solid tumours in this type of plastic. The seeding density was optimized for each cell line and was in the range of 2000-4000 cells/well. Heterogenous sensitivity to different cytotoxics was observed in the seven ovarian cancer cell lines tested in the ATP-based tumour chemosensitivity assay. The human ovarian carcinoma cell line, OVCA433, was found to be the most resistant cell line and 75% of the drugs showed an Index(SUM) above 300. Our results suggest that the use of appropriate culture conditions i.e. a serum-free culture environment, adherence-free growth and optimum seeding density can induce cell lines to behave more like tumour-derived cells in response to cytotoxic agents. On the basis of the comparison of chemosensitivity profiles of tumour-derived cells and cell lines derived from the corresponding tumour, a panel of cell lines can be selected. Such a panel could be used to screen and develop anticancer drugs.

The effect of imatinib mesylate (Glivec) on human tumor-derived cells.

Imatinib mesylate is a specific inhibitor of the Bcr-Abl protein tyrosine kinase that competes with ATP for its specific binding site in the kinase domain. It has activity against platelet-derived growth factor receptor alpha and beta (PDGFR-alpha and -beta), and c-kit, the receptor for stem cell factor. We have used a standardized ATP-tumor chemosensitivity assay and immunohistochemistry to determine the cytotoxicity of imatinib mesylate in tumor-derived cells from cutaneous and uveal melanoma, and ovarian carcinoma. Imatinib mesylate was tested at concentrations ranging from 2.0 to 0.0625 micromol/l alone and in combination with a cytotoxic drug (cisplatin, doxorubicin, paclitaxel or treosulfan). Imatinib mesylate showed low inhibition (IndexSUM>300) across the range of concentrations tested in this study, with few tumors exhibiting increasing inhibition with increased drug concentration. The median IC90 values for cutaneous and uveal melanoma and ovarian carcinoma were 13.2 micromol/l (4.0-294.3 micromol/l), 12.0 micromol/l (2.0-285.4 micromol/l) and 7.71 micromol/l (6.51-11.02 micromol/l), respectively. Imatinib mesylate potentiated the effect of different cytotoxics in 9% (5/54) of cases and had a negative effect in 13% (7/54) of cases, with no effect in the remainder. No correlation of effect was noted with c-kit, platelet-derived growth factor receptor-alpha or platelet-derived growth factor receptor-beta expression, assessed by immunohistochemistry. The signaling pathways mediated by activation of c-kit or platelet-derived growth factor receptor may act as antiapoptotic survival signals in some cancers and inhibition of these pathways may potentiate the activity of some cytotoxic drugs by inhibiting the survival signal. Growth inhibition, however, may reduce the efficacy of cytotoxic drugs, which tend to target proliferating cells preferentially, and clinical effects are therefore difficult to predict.

Pilot studies of the effect of zoledronic acid (Zometa) on tumor-derived cells ex vivo in the ATP-based tumor chemosensitivity assay.

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.

Cancer cell adaptation to chemotherapy.

BACKGROUND: Tumor resistance to chemotherapy may be present at the beginning of treatment, develop during treatment, or become apparent on re-treatment of the patient. The mechanisms involved are usually inferred from experiments with cell lines, as studies in tumor-derived cells are difficult. Studies of human tumors show that cells adapt to chemotherapy, but it has been largely assumed that clonal selection leads to the resistance of recurrent tumors. METHODS: Cells derived from 47 tumors of breast, ovarian, esophageal, and colorectal origin and 16 paired esophageal biopsies were exposed to anticancer agents (cisplatin; 5-fluorouracil; epirubicin; doxorubicin; paclitaxel; irinotecan and topotecan) in short-term cell culture (6 days). Real-time quantitative PCR was used to measure up- or down-regulation of 16 different resistance/target genes, and when tissue was available, immunohistochemistry was used to assess the protein levels. RESULTS: In 8/16 paired esophageal biopsies, there was an increase in the expression of multi-drug resistance gene 1 (MDR1) following epirubicin + cisplatin + 5-fluorouracil (ECF) chemotherapy and this was accompanied by increased expression of the MDR-1 encoded protein, P-gp. Following exposure to doxorubicin in vitro, 13/14 breast carcinomas and 9/12 ovarian carcinomas showed >2-fold down-regulation of topoisomerase IIalpha (TOPOIIalpha). Exposure to topotecan in vitro, resulted in >4-fold down-regulation of TOPOIIalpha in 6/7 colorectal tumors and 8/10 ovarian tumors. CONCLUSION: This study suggests that up-regulation of resistance genes or down-regulation in target genes may occur rapidly in human solid tumors, within days of the start of treatment, and that similar changes are present in pre- and post-chemotherapy biopsy material. The molecular processes used by each tumor appear to be linked to the drug used, but there is also heterogeneity between individual tumors, even those with the same histological type, in the pattern and magnitude of response to the same drugs. Adaptation to chemotherapy may explain why prediction of resistance mechanisms is difficult on the basis of tumor type alone or individual markers, and suggests that more complex predictive methods are required to improve the response rates to chemotherapy.

Rapid up-regulation of cyclooxygenase-2 by 5-fluorouracil in human solid tumors.

Inhibition of cyclooxygenase (COX)-2 has been associated with reduced growth of malignant cells. Current therapy of gastrointestinal carcinomas involves the use of 5-fluorouracil (5-FU)-based chemotherapy and we have therefore studied the effect of this agent on the expression of COX-2. COX-2 expression was measured by quantitative RT-PCR in biopsies from a series of 14 esophageal carcinomas, six of which had paired samples taken before and after chemotherapy, and in tumor-derived cells exposed to 5-FU in vitro from a series of 44 tumors, including breast, ovarian, esophageal and colonic carcinomas. COX-2 expression was increased by exposure to 5-FU or 5-FU combination chemotherapy in all the tumor types studied, whether measured in biopsies taken before and after 5-FU-based chemotherapy (4-fold increase, p<0.015) or in primary cells exposed to drugs in vitro (24-fold increase, p<0.001). A modest increase of COX-2 mRNA was also seen after in vitro treatment of cells with cisplatin. In contrast, doxorubicin and paclitaxel caused no up-regulation in vitro, while irinotecan caused inhibition of COX-2 (2.7-fold decrease, p<0.01). These data provide a molecular rationale for clinical trials of combination chemotherapy with COX-2 inhibitors.

The in vitro effect of gefitinib ('Iressa') alone and in combination with cytotoxic chemotherapy on human solid tumours.

BACKGROUND: Activation of the epidermal growth factor receptor (EGFR) triggers downstream signaling pathways that regulate many cellular processes involved in tumour survival and growth. Gefitinib ('Iressa') is an orally active tyrosine kinase inhibitor (TKI) targeted to the ATP-binding domain of EGFR (HER1; erbB1). METHODS: In this study we have used a standardised ATP-based tumour chemosensitivity assay (ATP-TCA) to measure the activity of gefitinib alone or in combination with different cytotoxic drugs (cisplatin, gemcitabine, oxaliplatin and treosulfan) against a variety of solid tumours (n = 86), including breast, colorectal, oesophageal and ovarian cancer, carcinoma of unknown primary site, cutaneous and uveal melanoma, non-small cell lung cancer (NSCLC) and sarcoma. The IC50 and IC90 were calculated for each single agent or combination. To allow comparison between samples the IndexSUM was calculated based on the percentage tumour growth inhibition (TGI) at each test drug concentration (TDC). Gefitinib was tested at concentrations ranging from 0.0625-2 microM (TDC = 0.446 microg/ml). This study represents the first use of a TKI in the assay. RESULTS: There was heterogeneity in the degree of TGI observed when tumours were tested against single agent gefitinib. 7% (6/86) of tumours exhibited considerable inhibition, but most showed a more modest response resulting in a low TGI. The median IC50 value for single agent gefitinib in all tumours tested was 3.98 microM. Interestingly, gefitinib had both positive and negative effects when used in combination with different cytotoxics. In 59% (45/76) of tumours tested, the addition of gefitinib appeared to potentiate the effect of the cytotoxic agent or combination (of these, 11% (5/45) had a >50% decrease in their IndexSUM). In 38% of tumours (29/76), the TGI was decreased when the combination of gefitinib + cytotoxic was used in comparison to the cytotoxic alone. In the remaining 3% (2/76) there was no change observed. CONCLUSION: The in vitro model suggests that gefitinib may have differential effects in response to concomitant cytotoxic chemotherapy with the agents tested during this study. The mechanism involved may relate to the effect of TKIs on growth rate versus their effect on the ability of the cell to survive the stimulus to apoptosis produced by chemotherapy.

Ex vivo characterization of XR11576 (MLN576) against ovarian cancer and other solid tumors.

XR11576 (MLN576) is a novel monophenazine with a mechanism of action that includes interaction with both topoisomerase (Topo) I and II. The aim of this study was to evaluate its cytotoxicity against fresh tumor cells taken from patients with a variety of solid tumors. Cells were obtained from 89 patients and exposed for 6 days to XR11576 alone, or in combination with doxorubicin, cisplatin, treosulfan, paclitaxel or vinorelbine. Cell survival was measured using the ATP-Tumor Chemosensitivity Assay (ATP-TCA). Immunohistochemical staining of Topo I, Topo IIalpha and MDR1 was performed on paraffin-embedded blocks in those tumors for which tissue was available (n = 49). Overall, the median IC90 and IC50 values of XR11576 in tumor-derived cells were 242 and 110 nM, respectively. In all samples XR11576 was more potent than the other cytotoxics tested. Breast and gynecological malignancies were most sensitive to XR11576, while the potency of this compound was slightly attenuated in gastrointestinal tumors, in which the median IC90 and IC50 values were 308 and 212 nM, respectively. Cases of synergism were identified when combining XR11576 with vinorelbine (nine of 30 samples) and doxorubicin (12 of 38 samples), while the addition of paclitaxel resulted in an antagonistic effect (CI50>1.2) in 38 of 42 tumors. A very modest correlation by linear regression analysis was found between the intensity of MDR1 staining and the IC50 of XR11576 (r = 0.311, p = 0.0312), but not with the IC90 (r = 0.247, NS). These data support the rapid introduction of XR11576 to clinical trials and suggest that it may be effective against a broad spectrum of tumor types.

Ex vivo reversal of chemoresistance by tariquidar (XR9576).

The expression of P-glycoprotein (P-gp) has been demonstrated to confer resistance to several anticancer drugs, including anthracyclines, taxanes and vinca alkaloids. Tariquidar is a novel inhibitor of P-gp that has been shown to reverse resistance to cytotoxic drugs in tumor cell lines and mouse xenografts. We have used an ATP-based chemosensitivity assay (ATP-TCA) to compare the activity of cytotoxic drugs in combination with tariquidar against a variety of solid tumors (n = 37). The expression of P-gp was determined in a subset of solid tumor samples by immunohistochemistry (n = 16). Resistance was seen in 20 of 37 (54%) tumors tested with doxorubicin, in 27 of 34 (79%) samples tested with paclitaxel and 17 of 31 (55%) with vinorelbine. Tariquidar alone showed no activity over a wide range of concentrations up to 2 microM (n = 14). The median IC90s for doxorubicin, paclitaxel and vinorelbine, alone were 2.57, 27.4 and 15.5 microM. These decreased to 1.67 (p<0.0005), 20.6 (p<0.05) and 9.5 microM (p<0.001), respectively, in combination with tariquidar. Tariquidar also significantly decreased resistance in 14 of 20 (70%), six of 27 (22%) and six of 17 (35%) samples tested with doxorubicin, paclitaxel and vinorelbine, respectively. Immunohistochemical staining for P-gp was positive in nine of 16 (56%) samples and in all of these cases addition of tariquidar improved the activity of the cytotoxic. The results show that tariquidar is able to decrease resistance in a number of solid tumors resistant to cytotoxic drugs known to be P-gp substrates. These data support the introduction of tariquidar in combination with chemotherapy to clinical trials of patients expressing P-gp.

The ex vivo characterization of XR5944 (MLN944) against a panel of human clinical tumor samples.

XR5944 (MLN944) is a novel DNA targeting agent with potent antitumor activity, both in vitro and in vivo, against several murine and human tumor models. We have used an ATP-tumor chemosensitivity assay to assess the ex vivo sensitivity of a variety of solid tumors (n = 90) and a CCRF-CEM leukemia cell line selected with XR5944. Differences in gene expression between the parental CCRF-CEM and the resistant subline were investigated by quantitative reverse transcription-PCR. Immunohistochemistry for topoisomerases I and IIalpha and multidrug resistance (MDR1) protein was done on those tumors for which tissue was available (n = 32). The CCRF-CEM XR5944 line showed increased mRNA levels of MDR1, major vault protein, and MDR-associated protein 1 compared with the parental line, whereas the expression of topoisomerases I, IIalpha, and IIbeta was essentially unchanged, suggesting that XR5944 is susceptible to MDR mechanisms. The median IC90 and IC50 values for XR5944 in tumor-derived cells were 68 and 26 nmol/L, respectively, 6-fold greater than in resistant cell lines. XR5944 was 40- to 300-fold more potent than the other cytotoxics tested, such as doxorubicin, topotecan, and paclitaxel. Breast and gynecologic malignancies were most sensitive to XR5944, whereas gastrointestinal tumors showed greater resistance. A positive correlation (r = 0.68; P < 0.0001) was found between the IC50 values of XR5944 and P-glycoprotein/MDR1 staining but not with either topoisomerase I or IIalpha immunohistochemistry index. These data support the rapid introduction of XR5944 to clinical trials and suggest that it may be effective against a broad spectrum of tumor types, especially ovarian and breast cancer.

Outcome of ATP-based tumor chemosensitivity assay directed chemotherapy in heavily pre-treated recurrent ovarian carcinoma.

BACKGROUND: We wished to evaluate the clinical response following ATP-Tumor Chemosensitivity Assay (ATP-TCA) directed salvage chemotherapy in a series of UK patients with advanced ovarian cancer. The results are compared with that of a similar assay used in a different country in terms of evaluability and clinical endpoints. METHODS: From November 1998 to November 2001, 46 patients with pre-treated, advanced ovarian cancer were given a total of 56 courses of chemotherapy based on in-vitro ATP-TCA responses obtained from fresh tumor samples or ascites. Forty-four patients were evaluable for results. Of these, 18 patients had clinically platinum resistant disease (relapse < 6 months after first course of chemotherapy). There was evidence of cisplatin resistance in 31 patients from their first ATP-TCA. Response to treatment was assessed by radiology, clinical assessment and tumor marker level (CA 125). RESULTS: The overall response rate was 59% (33/56) per course of chemotherapy, including 12 complete responses, 21 partial responses, 6 with stable disease, and 15 with progressive disease. Two patients were not evaluable for response having received just one cycle of chemotherapy: if these were excluded the response rate is 61%. Fifteen patients are still alive. Median progression free survival (PFS) was 6.6 months per course of chemotherapy; median overall survival (OAS) for each patient following the start of TCA-directed therapy was 10.4 months (95% confidence interval 7.9-12.8 months). CONCLUSION: The results show similar response rates to previous studies using ATP-TCA directed therapy in recurrent ovarian cancer. The assay shows high evaluability and this study adds weight to the reproducibility of results from different centres.

Heterogeneity of chemosensitivity of esophageal and gastric carcinoma.

Esophageal and gastric cancer have a poor prognosis, and chemotherapy is rarely of long-term benefit. This may be related in part to heterogeneity of chemosensitivity and to constitutive resistance to individual cytotoxic drugs. This study aimed to demonstrate the degree of heterogeneity of chemosensitivity between tumors. We have examined the heterogeneity of chemosensitivity in esophageal and gastric cancer specimens (n=85) using an ex vivo ATP-based chemosensitivity assay (ATP-TCA). A variety of chemotherapeutic agents were tested. Sixty-four specimens were endoscopic biopsy samples; the remainder were from resection specimens. Cells were obtained from 62 specimens (73%). Eight assays were infected due to contamination/infection of the biopsy material, giving an evaluability rate of 87%. Analysis of the data showed considerable heterogeneity of chemosensitivity. The most active single agents identified by the assay were mitomycin C (56% sensitivity) and 5-fluorouracil (5-FU; 42% sensitivity). Exposure of tumor cells to combinations of drugs showed ECF (epirubicin, cisplatin, 5-FU) and mitomycin C+5-FU to be moderately active regimens. Other experimental drug combinations showed greater activity. There is a marked heterogeneity of chemosensitivity in esophageal and gastric cancers. The degree of heterogeneity observed suggests that the ATP-TCA could be used to individualize chemotherapy by selecting agents for particular patients. This approach provides the rationale for a trial of ATP-TCA-directed therapy to determine whether individualization of chemotherapy might improve patient response and survival.

Heterogeneity of chemosensitivity of colorectal adenocarcinoma determined by a modified ex vivo ATP-tumor chemosensitivity assay (ATP-TCA).

Advanced colorectal cancer (CRC) has a poor prognosis with a 5-year survival of only 5% despite treatment with chemotherapeutic agents. Response rate and overall survival varies little between the commonly used single agents, although combinations achieve better outcomes. It is well established that considerable heterogeneity exists between cancers of the same tissue type, but it has been difficult to establish this for CRC. We therefore investigated the heterogeneity of chemosensitivity in CRC using a modified version of the ex vivo ATP-tumor chemosensitivity assay (ATP-TCA) capable of handling infected tumor tissue. Fifty-three specimens of primary solid or malignant effusions of CRC were tested, of which 46 (87%) were evaluable. There were considerable differences in sensitivities between individuals. The most active single cytotoxic agents in the assay were identified as 5-fluorouracil, irinotecan and mitomycin C (MMC). Cells were exposed to combinations of drugs added simultaneously at the same concentrations tested as single agents. All drug combinations achieved greater growth inhibition than drugs used alone. MMC+gemcitabine was found to be the most effective combination in 83% of specimens. The ATP-TCA has previously been shown to be a good predictor of response to chemotherapy in other tissue types. The degree of heterogeneity demonstrated from these results suggests that the ATP-TCA could be used to identify patients who might benefit from specific chemotherapeutic agents alone or in combination.

Use of an ATP-based chemosensitivity assay to design new combinations of high-concentration doxorubicin with other drugs for recurrent ovarian cancer.

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.