Ten-year results of intense dose-dense chemotherapy show superior survival compared with a conventional schedule in high-risk primary breast cancer: final results of AGO phase III iddEPC trial.

Background: Primary breast cancer (BC) patients with extensive axillary lymph-node involvement have a limited prognosis. The Arbeitsgemeinschaft fuer Gynaekologische Onkologie (AGO) trial compared intense dose-dense (idd) adjuvant chemotherapy with conventionally scheduled chemotherapy in high-risk BC patients. Here we report the final, 10-year follow-up analysis. Patients and methods: Enrolment took place between December 1998 and April 2003. A total of 1284 patients with 4 or more involved axillary lymph nodes were randomly assigned to receive 3 courses each of idd sequential epirubicin, paclitaxel and cyclophosphamide (iddEPC) q2w or standard epirubicin/cyclophosphamide followed by paclitaxel (EC â†’ P) q3w. Event-free survival (EFS) was the primary end point. Results: A total of 658 patients were assigned to receive iddEPC and 626 patients were assigned to receive EC â†’ P. The median duration of follow-up was 122 months. EFS was 47% (95% CI 43% to 52%) in the standard group and 56% (95% CI 52% to 60%) in the iddEPC group [hazard ratio (HR) 0.74, 95% CI 0.63-0.87; log-rank P = 0.00014, one-sided]. This benefit was independent of menopausal, hormone receptor or HER2 status. Ten-year overall survival (OS) was 59% (95% CI 55% to 63%) for patients in the standard group and 69% (95% CI 65% to 73%) for patients in the iddEPC group (HR = 0.72, 95% CI 0.60-0.87; log-rank P = 0.0007, two-sided). Nine versus two cases of secondary myeloid leukemia/myelodysplastic syndrome were observed in the iddEPC and the EC â†’ P arm, respectively. Conclusion: The previously reported OS benefit of iddEPC in comparison to conventionally dosed EC â†’ P has been further increased and achieved an absolute difference of 10% after 10 years of follow-up.

Plasma and tissue pharmacokinetics of epirubicin and Paclitaxel in patients receiving neoadjuvant chemotherapy for locally advanced primary breast cancer.

The objective of the study was to assess individual distribution of antineoplastic drugs into the tumor. Twelve advanced-stage primary breast cancer patients with neoadjuvant epirubicin+paclitaxel chemotherapy were studied. Plasma concentrations of epirubicin and paclitaxel were monitored for 24 h. Epirubicin concentrations in subcutaneous and tumor tissues were measured using microdialysis up to 12 h postdose. Epirubicin concentrations were described by a compartmental population pharmacokinetic model (NONMEM). Noncompartmental analysis was used for paclitaxel. Plasma pharmacokinetics corresponded to published data. Mean epirubicin exposure in the tumor and in subcutaneous tissue was very similar, but tissue Cmax and area under the curve values reached only (means) 1% and 11%, respectively, of plasma values. Epirubicin doses were significantly correlated to tumor exposure irrespective of body surface area. There is no specific barrier for epirubicin to reach primary breast cancer tumors.

Preeclamptic women are deficient of interleukin-10 as assessed by cytokine release of trophoblast cells in vitro.

BACKGROUND: It is well known that the acceptance of the fetoplacental unit in human pregnancy requires maternal immune tolerance, which is thought to be regulated locally by the placenta. Therefore an anti-inflammatory cytokine such as IL-10 plays a critical role in different pregnancy disorders including preeclampsia. In the present study, we examined the expression of both proinflammatory (TNF-alpha, IL-1beta, IL-2) and immunoregulatory (IL-6, IL-10) cytokines from normal term and preeclamptic patients in human trophoblast cultures. METHODS: Eleven patients with preeclampsia and 11 patients with a normal pregnancy at term were included in the study. Trophoblast cells isolated from placentas were cultured up to 48 h under standard tissue culture conditions and cytokine release was determined by ELISA. IL-10 synthesis was significantly decreased in the third trimester in preeclamptic patients in comparison with the control group. RESULTS: There were no significant differences in IL-1beta, IL-2, IL-6 or TNF-alpha expression but a significant alteration in IL-10 release in trophoblast cultures in vitro in term placentas from preeclamptic patients compared with normal pregnancy. CONCLUSIONS: Because IL-10 is a potent regulator of anti-inflammatory immune response these abnormalities may be associated with the inadequate placental development in preeclampsia.

Chemosensitivity testing in gynecologic oncology--dream or reality?

Cell culture and animal models have played an essential role in the research of new principles of therapy. Many methods for the individualized testing of therapy sensitivity and resistance have been developed, for example, the clonogenic assay. Presently, the ATP-TCA is commercially available as a testing kit. This review gives an overview of the tumor samples that were tested in the oncologic laboratory in the Department of Obstetrics and Gynecology, Munich Grosshadern between 1993 and 2001. All target parameters show a clear trend in favor of sequential, dose-intensified Epirubicin/Paclitaxel therapy. If this trend remains valid for the total number of patients, a significant impact of this new principle of therapy can be expected. By individualized planning of therapy with ATP-TCA testing, therapy in the individual patient could already be performed by the examination of sensitivity in the preoperative biopsy specimen.

Weekly carboplatin and docetaxel for locally advanced primary and recurrent cervical cancer: a phase I study.

OBJECTIVE: The aim of this study was to determine the dose-limiting toxicities (DLTs) and maximum tolerated doses (MTDs) of a docetaxel-carboplatin regimen in patients with locally advanced cervical cancer (LACC) or recurrent cervical cancer. The regimen was administered weekly, with a maximum of 12 courses. PATIENTS AND METHODS: Twenty patients were treated with with a total of 145 cycles of weekly carboplatin and docetaxel. The starting dose of docetaxel was 25 mg/m(2) with increments of 5 mg/m(2) until a final dose of 35 mg/m(2) was reached. Dose-escalation of docetaxel was followed by carboplatin at AUC 2, AUC 2.5, and AUC 3, respectively. Defined dose-limiting toxicities were WHO grade (G) 3 hematotoxicity, G4 mucositis, and G2 neurotoxicity. The response status of the patients was assessed using the common ECOG response criteria. RESULTS: Two of four patients developed a DLT at dose level 4. Nonhematological toxicity was generally mild, except for ubiquitous complete alopecia. The MTD was reached at docetaxel 35 mg/m(2) and carboplatin AUC 2 mg/mL.min. The overall response rate was 65% in the entire group of evaluable patients and 77% in patients with primary LACC, with two cases of pathological complete response. CONCLUSION: This dose-dense regimen was well-tolerated and could be administered on an outpatient basis.

The role of chemotherapy in invasive cancer of the cervix uteri: current standards and future prospects.

For many decades, invasive cervical cancer has been considered more or less chemoresistant and chemotherapy has been limited to patients presenting with overt metastatic disease or those suffering from pelvic recurrences which could not be advised to secondary local treatments. However, more than 20 different single agents are considered active in cervical cancer. Recent cooperative clinical trials have demonstrated the superiority of multi-modality strategies for patients with high-risk cervical cancer. These studies integrating chemotherapy as part of the primary therapeutic concept have provided the most significant improvement of locally advanced disease in more than three decades. This review summarizes current standards of chemotherapy for invasive cervical cancer and shows new developments which may improve systemic treatment of the disease.

Interaction of cisplatin, paclitaxel and adriamycin with the tumor suppressor PTEN.

Due to its pivotal role in signal transduction, the universal tumor suppressor PTEN (also termed MMAC or TEP) is one of the putative candidates for involvement in tumorigenesis of several tissues. Although involvement of PTEN in tumorigenesis was shown in different tissues, no data are available concerning PTEN activity in response to antineoplastic agents. Therefore, we assayed the PTEN activity exposed to either blank medium or the commonly used anti-cancer drugs cisplatin, adriamycin or paclitaxel, respectively, in three different concentrations. PTEN activity was determined using the Malachite Green assay basing upon dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3) by the PTEN enzyme and subsequent determination of inorganic phosphate released. Although the three different anti-cancer drugs assayed act with different cellular modes, the antineoplastics influenced PTEN activity in a similar manner: at low concentrations tested all three antineoplastics significantly increased PTEN activity. However, increasing drug concentrations exhibited a decline but not a total loss of PTEN activity. The data indicate that PTEN activity is increased following cytotoxic drug exposure and, thereby, exhibits its suppressive function. However, the decrease of PTEN activity in response to increasing drug concentrations suggests an aberration of total functional activity. As far as the regulative checkpoint PTEN is abolished, tumor cells might evade cell death pathways resulting in increased proliferation of cancer cells. This might be a general event in refractory tumor cells surviving chemotherapy.

Comparison of the ex vivo chemosensitivity of uveal and cutaneous melanoma.

Cutaneous and uveal melanoma both have a poor prognosis and chemotherapy is usually unsuccessful. We have previously reported the activity of a number of cytotoxic agents against metastatic cutaneous and primary choroidal uveal melanoma using an ex vivo adenosine triphosphate (ATP)-based chemosensitivity assay (ATP-TCA). In this study we compare the results obtained with the two types of melanoma. Cutaneous melanoma deposits in skin and lymph nodes (n = 58) and choroidal melanomas (n = 77) were tested using the ATP-TCA. Analysis of the data based on an arbitrary threshold for sensitivity shows that both types of melanoma exhibit heterogeneity of sensitivity to all the agents and combinations tested. With all the single agents except gemcitabine, cutaneous melanomas showed greater sensitivity in the assay, though this did not achieve statistical significance. This was also true with the drug combinations, with the exception of treosulfan + gemcitabine, which had similar activity in each type of melanoma. Of all the single agents tested, doxorubicin (47% of specimens classed as sensitive), vinorelbine (43%), treosulfan (41%) and paclitaxel (33%) showed the greatest activity with cutaneous melanoma. In the uveal melanoma samples, mitoxantrone (33%), gemcitabine (22%) and treosulfan (21%) showed the greatest activity. In contrast to the cutaneous melanomas, 13% of the uveal melanomas were sensitive to paclitaxel, 4% were sensitive to doxorubicin and 11% were found to be sensitive to vinorelbine. Both tumour types showed greater sensitivity to combinations of cytotoxic agents. The combination of treosulfan + gemcitabine was universally effective, with 72% of cutaneous melanomas and 80% of uveal melanomas exhibiting activity at the level selected to indicate sensitivity in the assay, though this will not necessarily indicate a similar level of clinical sensitivity.

Prognostic value of repeated serum CA 125 measurements in first trimester pregnancy.

OBJECTIVE: To assess the diagnostic value of maternal CA 125 in patients with symptomatic first trimester pregnancy and to evaluate the prognostic significance of CA 125 versus beta-hCG in early pregnancies with intact fetal heartbeat, complicated by vaginal bleeding. STUDY DESIGN: Two prospective open-label studies with longitudinal follow-up in the second trial. SETTING: Academic Department of Obstetrics and Gynecology, University of Cologne. PATIENTS: Study 1: 168 patients presenting between gestational weeks 6 and 12 with: extrauterine pregnancy, 29; missed abortion, 50; incomplete spontaneous abortion, 38; imminent abortion, 33; and normal pregnancy (no history of endometriosis or ovarian mass), 18. Study 2: Fifty consecutive patients with vaginal bleeding during gestational weeks 6-12 all of whom having demostrable fetal heartbeat. Eighteen patients finally aborted whereas the remainder had normally continuing pregnancy until term. MAIN OUTCOME MEASURE: Study 1: Single serum determinations of CA 125 and beta-hCG were correlated with the different disorders observed. Study 2: Two sequential measurements of serum CA 125 and beta-hCG performed within a 5-7 days interval were related to the outcome of pregnancy as indicated by changes of the ultrasound presentation, miscarriage, future hospitalization, or delivery. RESULTS: Study 1: Patients with vaginal bleeding generally had higher median CA 125 values (38 IU/ml; range 1.3-540) compared to non-bleeding patients (17.8 IU/ml; range 1.0-157). No statistically significant differences in regard to median serum CA 125 levels between symptomatic and normal pregnancies occurred: normal pregnancy, 25.5 IU/ml (range 3.2-97); ectopic pregnancy, 26 IU/ml (range 1.3-157); missed abortion, 19.1IU/ml (range 1-242); threatened abortion, 48 IU/ml (range 5.2-540); spontaneous abortion, 40 IU/ml (range 5.4-442). Study 2: Initial CA 125 levels did not differ significantly between both groups of patients with 27/32 non-aborters and 13/18 aborters showing concentrations below 65 IU/ml. After 5-7 days, CA 125 in all patients who eventually aborted remained high or increased whereas non-aborters all had constantly low or steeply declining CA 125 measures. beta-hCG increased in all non-aborters but also in 13/18 aborters during the 5-7 day interval. CONCLUSION: Single serum measurements of CA 125 in symptomatic first trimester pregnant patients failed to discriminate spontaneous abortion, ectopic or normal pregnancies. However, sequential determinations of maternal CA 125 measurements appear to be a highly sensitive prognostic marker in patients with viable pregnancy at risk for abortion.

Heterogeneity of proteinkinase C activity and PKC-zeta expression in clinical breast carcinomas.

Proteinkinase C (PKC) is involved in carcinogenesis, proliferation, and metastatic spread of breast cancer. New anticancer strategies have been developed with PKC as a potential target for therapeutic intervention. However, most of the encouraging preliminary data were observed in breast cancer cell lines only. Insignificant information is available concerning clinical breast cancer cells. Our aim was to investigate the involvement of PKC in clinical breast carcinoma cells. To this end, we set up short-term cultures (3 days) of native tumor cells derived from 12 patients with advanced breast cancer. Addition of commonly used antineoplastics, including both single agents and combinations (tamoxifen, Adriamycin, paclitaxel, Adriamycin plus paclitaxel, epirubicin plus 4-OOH-cyclophosphamide, mitoxantrone, mitoxantrone plus vinorelbin, vinorelbin), simulated the clinical situation. In relation to each control we determined total PKC activity and quantified the PKC-zeta isoform. In 6 patients, no obvious alteration of PKC activities was detected. In the remainder, either inhibition or augmentation of PKC activity in the presence of cytostatics was detected. However, no tendency could be observed concerning the influence of the therapeutics on PKC activity. PKC-zeta expression was much more heterogeneous than activity assays. Although anticancer drugs influenced PKC-zeta expression, the results showed no uniformity with regard to PKC-zeta expression. Moreover, PKC-zeta expression did not correlate with total PKC activity, indicating a differential expression of different PKC isoenzymes. Therefore, we conclude that both PKC activity and PKC-zeta expression differ individually. More data concerning this topic are necessary prior to offering a clinically useful PKC-tailored regimen.

Detection of aberrations of chromosome 17 and p53 gene expression and their correlation to histologic grading and prognosis in primary invasive squamous cell carcinoma of the cervix.

We analyzed tumor tissues from 14 patients with invasive squamous cell carcinoma of the cervix for aberrations of chromosome 17 and p53 expression. All but 3 patients were negative for p53 protein expression, the protein being detected in 2 International Federation of Obstetrics and Gynecology stage IIa cancers and 1 stage Ib G3 carcinoma. Significant cytogenetic aberrations in the form of losses and gains of chromosome 17 were diagnosed in 9 and 7 patients, respectively. There was no correlation with tumor prognosis, clinical stage or histologic grade. According to most reports, almost all cervical carcinomas contain integrated human papilloma virus (HPV) and express E6 oncoproteins. Increasing evidence suggests that E6 protein interaction leads to p53 mutation in HPV-infected cervical epithelium. Since most cervical tumors are infected with HPV, and the tumors originate through p53 gene mutation caused by the said interaction, which leads subsequently to the overexpression of p53 oncoprotein, lack of the latter in the remaining 11 cervical tumors may either be the result of technical shortcomings, or the tumor may arise in such circumstances through a p53-independent pathway. On the other hand, 2 of 3 stage IIa cancers and 1 Ib G3 carcinoma were found to be p53 positive, thus supporting the notion that p53 inactivation is a relatively late event in the progression of cervical cancer.

Liposomal doxorubicin and weekly paclitaxel in the treatment of metastatic breast cancer.

The combination of paclitaxel and doxorubicin or epirubicin is highly active against metastatic breast cancer, yet may produce congestive heart failure. Liposome-encapsulated doxorubicin is a new formulation of doxorubicin with no dose-limiting cardiac toxicity. Twenty-one patients with metastatic breast cancer were treated with pegylated liposomal doxorubicin (20 mg/m2, day 1) and paclitaxel (100 mg/m2, days 1 and 8) for six cycles every 2 weeks. All patients had had relapse or progression on one to five previous chemotherapies. We observed two patients with complete and eight patients with partial remissions (48% response rate). Eight of the 10 responders had had previous therapy with epirubicin, doxorubicin or mitoxantrone. The mean remission duration was 5 months. Disease progression due to brain metastasis occurred in five cases. Severe side effects (grade 3 WHO) were alopecia (100%), skin toxicity in 29%, neuropathy in 24% and mucositis in 13%. Leukopenia (grade 4 WHO) was observed in 48%, but there was no cardiac toxicity, no death and no hospitalization. The combination of weekly paclitaxel and liposomal doxorubicin every 2 weeks is highly effective in previously treated patients. Based on the doses we administered, we recommend 15 mg/m2 liposomal doxorubicin every 2 weeks and 80 mg/m2 paclitaxel weekly.

Intra-arterial mitoxantrone and paclitaxel in a patient with Stewart-Treves syndrome: selection of chemotherapy by an ex vivo ATP-based chemosensitivity assay.

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.

Correlation of drug response with the ATP tumorchemosensitivity assay in primary FIGO stage III ovarian cancer.

OBJECTIVE: Our purpose was to: (a) study the in vitro chemosensitivity of primary epithelial ovarian cancer to drug combinations with cisplatin (CDDP), carboplatin (CBDCA), paclitaxel (PTX), epirubicin (EPI), or cyclophosphamide (CTX) utilizing the ATP tumorchemosensitivity assay (ATP-TCA); (b) correlate the test results with clinical response in patients with FIGO stage III ovarian cancer; and (c) analyze the most useful parameters for interpretation of test results. METHODS: CBDCA/CTX, CBDCA/PTX, CDDP/PTX, and EPI/PTX were tested in 93 fresh human primary epithelial ovarian cancer specimens. Correlations of in vitro drug sensitivity/resistance and clinical response were performed in 38 patients with FIGO stage III disease utilizing Fisher's exact test and by comparison of progression-free (PFS) and overall survival (OS) between those testing as sensitive or resistant. A progression-free interval of more than 12 months following surgery was classified as clinical response. ATP-TCA results were analyzed using the median effective dose, area under the curve, or a defined sensitivity index. RESULTS: Evaluable test results were achieved in 83 of 93 patients (89%). EPI/PTX had the highest in vitro activity (P < 0.001). In the clinical correlation, 29 of 38 patients (76%) were classified as in vitro sensitive (sensitivity index [SI] <250) and 9 patients as in vitro resistant (SI >250). The SI was superior for interpretation of test results. Patients testing as chemosensitive had a significantly longer mean PFS (28.5 vs 12.6 months, P = 0.033) and OS (46.1 vs 17.6, P = 0.03) compared to those patients predicted to be resistant. The assay demonstrated a sensitivity, specificity, and positive and negative predictive value of 95, 44, 66, and 89%, respectively (Fisher's exact test, P = 0. 007). CONCLUSION: The observed in vitro efficacy of EPI/PTX in primary epithelial ovarian cancer specimens warrants further clinical evaluation. The high evaluability rate and the observed correlation with PFS and OS, within the limitations of a nonrandomized study, support the use of the ATP chemosensitivity assay in future prospective assay-directed trials.

Anti-neoplastic activity of topotecan versus cisplatin, etoposide and paclitaxel in four squamous cell cancer cell lines of the female genital tract using an ATP-Tumor Chemosensitivity Assay.

We evaluated the in vitro cytotoxicity of topotecan (TPT), versus cisplatin, etoposide (VP-16) and paclitaxel (PTX) in four squamous cell cancer cell lines of the cervix uteri and vulva. Four established human squamous cancer cell lines from the cervix uteri (A-431, Ca Ski and C-33) and vulva (CAL-39) were used. The cytotoxic effects of the agents were examined using the ATP-Tumor Chemosensitivity Assay (ATP-TCA). In addition to the single agents, the following combinations were tested: TPT+cisplatin, TPT+VP-16 and TPT+PTX. Three cell lines (C-33, Ca Ski and CAL-39) were highly sensitive to TPT, but one cell line (A-431) was less sensitive. Furthermore, the cytotoxic activity of TPT was superior to that of cisplatin in Ca Ski and C-33 cells, but inferior in CAL-39 and A-431. TPT was also more active than VP-16 in CAL-39 and Ca Ski. On the other hand, the cytotoxic activity of TPT was weaker than PTX in C-33, CAL-39 and A-431. TPT increased the cytotoxic activity of cisplatin and VP-16 in C-33, Ca Ski and A-431. However, synergistic features were observed only in A-431 cells. TPT also enhanced the cytotoxic activity of PTX in A-431 and Ca Ski. In CAL-39 and C-33, however, increased cytotoxic activity occurred only at higher drug concentrations, whereas antagonism was observed at lower drug concentrations. In conclusion, our results suggest that TPT has a significant cytotoxic effect on most squamous cell cancer cell lines which may be superior to cisplatin, VP-16 and PTX in some instances. Furthermore, TPT is likely to potentiate the cytotoxic activity of these agents in individual cell lines tested.

The ex vivo effect of high concentrations of doxorubicin on recurrent ovarian carcinoma.

The cardiotoxicity of anthracyclines has largely prevented dose intensification, but the use of liposomal preparations (e.g. Caelyx/Doxil) allows much higher intra-tumoral concentrations to be achieved without cardiotoxicity. However, it is uncertain how much this will improve response rates over standard anthracycline therapy. The ATP-based chemosensitivity assay (ATP-TCA) has been used to develop new regimens for several tumor types, to investigate the molecular basis of chemosensitivity and shows considerable promise as a clinical method for individualizing chemotherapy. In this study, we have used the ATP-TCA to determine the concentration responsiveness of tumor-derived cells to concentrations of doxorubicin. The 22 tumor samples included were obtained from 20 heavily pretreated patients with recurrent ovarian cancer. Eight had previous anthracycline exposure, four as part of the CAP regimen. The results show more than 95% inhibition at clinically achievable concentrations in 11 of 22 tumors tested. Of the rest, seven showed a plateau effect between 80 and 95% inhibition, suggesting that there might be a subset of resistant cells present that is not inhibited by high concentrations of doxorubicin. Two tumors showed complete resistance and neither of these had previously received anthracycline therapy. As it has been suggested that gemcitabine might enhance anthracycline sensitivity in combination and we have had good results with gemcitabine modulation of alkylating agents in the assay, we have tested the combination of doxorubicin+gemcitabine under assay conditions in 11 tumors with little indication of improvement. In conclusion, doxorubicin at concentrations achievable with liposomal preparations shows strong ex vivo activity against pretreated recurrent ovarian cancer in just over half of the cases tested.

Lack of correlation between P53 expression, BCL-2 expression, apoptosis and ex vivo chemosensitivity in advanced human breast cancer.

The relationship between apoptosis and chemosensitivity remains complex. We tested the chemosensitivity of 45 patients with advanced breast cancer (BC) ex vivo against anthracyclines (A: doxorubicin, epirubicin), taxanes (T: paclitaxel, docetaxel), cisplatin (DDP) and CMF and any correlation with the expression of p53, Bcl-2 and apoptosis. Viable cells were processed for ex vivo ATP Tumor Chemosensitivity Assay (ATP-TCA). Immunohistochemistry was performed in corresponding tumor samples. Apoptosis prior to chemotherapy was assayed using a TUNEL Test. Of 45 BC tested, 18 (40%) were p53+ and 37 (82%) showed high Bcl-2 expression. Apoptosis was detected in 29 (64.4%) specimens. The Ex vivo Response Rate (EVRR) for T was 75.6% in all cases. This was the highest rate among the 4 drugs tested followed by CMF (66.7%). For A and DDP the positive rates were lower (27.6% and 10.6%, respectively). A significant correlation (r = 0.589, p < or = 0.01) was found between tumors which were sensitive to A and DDP. There was no association between chemosensitivity and apoptosis. Moreover tests for p53 and Bcl-2 did not show a correlation to ex vivo chemosensitivity. Pretreatment apoptotic parameters are unlikely to predict the individual response of breast cancer to antineoplastic agents.

Ex vivo assays to evaluate the role of protein kinase C in tumor cells of patients with breast cancer.

New anti-cancer strategies have been developed with respect to proteinkinase C (PKC) as a potential target for therapeutic intervention in patients with advanced breast cancer. Using cell lines, most of the preliminary data are encouraging but insufficient information is available concerning clinical breast cancer cells. Thus, we decided to clarify the involvement of PKC in clinical breast carcinoma cells. We isolated viable tumor cells from fluids or tissue burden of eleven patients with advanced breast cancer. Performance of short term cultures supplemented with commonly used antineoplastics mimicked the clinical situation. We determined the ex vivo chemosensitivity pattern of each cell population. Additionally, we analysed total PKC activity and quantified the PKC-isoform eta. All assays showed a heterogeneous highly variable distribution of the data investigated. No tendency could be observed regarding the influence of the therapeutics on PKC activity, PKC-eta expression or chemoresistance, respectively. Moreover, changes in neither PKC-eta expression, PKC activity nor chemoresistance induced by a particular drug in an individual tumor necessarily predicted the same reaction in another tumor to this agent. Therefore, we concluded that more explorative data concerning this topic are required prior to the development of a clinically useful therapy regimen with PKC as the major target.

ATP-based tumor chemosensitivity testing: assisting new agent development.

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.

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.