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Background: The phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways are frequently activated in breast cancer which can result in antioestrogen resistance. Single protein markers failed to be introduced into clinical practice. We, therefore, aimed to find a better read-out of activation of the PI3K and MAPK pathways in ER+/HER2- breast cancer. Assessment of seven PI3K/MAPK proteins might better reflect pathway activation and distinguish patients without adjuvant tamoxifen benefit., Methods: Tumour blocks were recollected from 293 primary postmenopausal ER+/HER2- breast cancer patients randomised between tamoxifen and no adjuvant therapy. PTEN, p-AKT(Thr308), p-AKT(Ser473), p-p70S6K, p-4EBP1, p-ERK1/2 and p-S6RP expression was assessed by immunohistochemistry followed by unsupervised hierarchical clustering. The primary endpoint was recurrence-free interval. Multivariate Cox models were used to assess tamoxifen benefit. A classification tool was developed based on protein expression profile., Results: Subgroups were identified with preferentially activated (A) and preferentially not activated (N) proteins. Patients in group N derived significant benefit from tamoxifen (multivariate hazard ratio (HR) = 0.23, p = 0.000101), while patients from group A did not (multivariate HR = 1.37, p = 0.64), p for interaction 0.020. Our generated classification tool confirmed these results (p for interaction 0.024)., Conclusions: Hierarchical clustering of seven PI3K/MAPK proteins reflects pathway activation and can guide treatment decisions in primary ER+/HER2- postmenopausal breast cancer patients.

Background: The purpose of this study was to evaluate single-nucleotide polymorphisms (SNPs) in genes encoding key metabolising enzymes or involved in pharmacodynamics for possible associations with paclitaxel-induced peripheral neuropathy., Methods: The study population consists of 188 women from the multicenter, randomised, phase II ATX trial (BOOG2006-06; EudraCT number 2006-006058-83) that received paclitaxel and bevacizumab without or with capecitabine as first-line palliative therapy of HER2-negative metastatic breast cancer. Genotyping of CYP2C8*3 (c.416G>A), CYP3A4*22 (c.522-191C>T), TUBB2A (c.-101T>C), FGD4 (c.2044-236G>A) and EPHA5 (c.2895G>A) was performed by real-time PCR. Toxicity endpoints were cumulative dose (1) until first onset of grade ⩾1 peripheral neuropathy and (2) until first paclitaxel dose reduction from related toxicity (NCI-CTCAE version 3.0). SNPs were evaluated using the Kaplan-Meier method, the Gehan-Breslow-Wilcoxon test and the multivariate Cox regression analysis., Results: The rate of grade ⩾1 peripheral neuropathy was 67% (n=126). The rate of dose reduction was 46% (n=87). Age ⩾65 years was a risk factor for peripheral neuropathy (HR=1.87, P<0.008), but not for dose reduction. When adjusted for age, body surface area and total cumulative paclitaxel dose, CYP2C8*3 carriers had an increased risk of peripheral neuropathy (HR=1.59, P=0.045). FGD4 c.2044-236 A-allele carriers had an increased risk of paclitaxel dose reduction (HR per A-allele=1.38, P=0.036) when adjusted for total cumulative paclitaxel dose., Conclusions: These findings may point towards clinically useful indicators of early toxicity, but warrant further investigation.

Background: Only a small proportion of patients respond to anti-VEGF therapy, pressing the need for a reliable biomarker that can identify patients who will benefit. We studied the biological activity of anti-VEGF antibodies in patients' blood during anti-VEGF therapy by using the Ba/F3-VEGFR2 cell line, which is dependent on VEGF for its growth., Methods: Serum samples from 22 patients with cancer before and during treatment with bevacizumab were tested for their effect on proliferation of Ba/F3-VEGFR2 cells. Vascular endothelial growth factor as well as bevacizumab concentrations in serum samples from these patients were determined by enzyme linked immunosorbent assay (ELISA)., Results: The hVEGF-driven cell proliferation was effectively blocked by bevacizumab (IC 50 3.7 μg ml -1 ; 95% CI 1.7-8.3 μg ml -1 ). Cell proliferation was significantly reduced when patients' serum during treatment with bevacizumab was added (22-103% inhibition compared with pre-treatment). Although bevacizumab levels were not related, on-treatment serum VEGF levels were correlated with Ba/F3-VEGFR2 cell proliferation., Conclusions: We found that the neutralising effect of anti-VEGF antibody therapy on the biological activity of circulating VEGF can be accurately determined with a Ba/F3-VEGFR2 bioassay. The value of this bioassay to predict clinical benefit of anti-VEGF antibody therapy needs further clinical evaluation in a larger randomised cohort.

Background: As a prelude to combination studies aimed at resistance reversal, this dose-escalation/dose-expansion study investigated the selective Src kinase inhibitor saracatinib (AZD0530) in combination with carboplatin and/or paclitaxel., Methods: Patients with advanced solid tumours received saracatinib once-daily oral tablets in combination with either carboplatin AUC 5 every 3 weeks (q3w), paclitaxel 175 mg m(-2) q3w, paclitaxel 80 mg m(-2) every 1 week (q1w), or carboplatin AUC 5 plus paclitaxel 175 mg m(-2) q3w. The primary endpoint was safety/tolerability., Results: A total of 116 patients received saracatinib 125 (N=20), 175 (N=44), 225 (N=40), 250 (N=9), or 300 mg (N=3). There were no clear dose-related trends within each chemotherapy regimen group in number or severity of adverse events (AEs). However, combining all groups, the occurrence of grade ≥3 asthenic AEs (all causality) was dose-related (125 mg, 10%; 175 mg, 20%; ≥225 mg, 33%), and grade ≥3 neutropenia occurred more commonly at doses ≥225 mg. There was no evidence that saracatinib affected exposure to carboplatin or paclitaxel, or vice versa. Objective responses were seen in 5 out of 44 patients (11%) receiving carboplatin plus paclitaxel q3w, and 5 out of 24 (21%) receiving paclitaxel q1w., Conclusion: Saracatinib doses up to 175 mg with paclitaxel with/without carboplatin showed acceptable toxicity in most patients, and are suitable for further trials., (© 2012 Cancer Research UK)

Background: Sunitinib is a multitargeted, oral tyrosine kinase inhibitor with antitumour and antiangiogenic activity. We investigated the safety and pharmacokinetics of sunitinib in combination with irinotecan in patients with advanced, refractory solid tumours., Methods: Sunitinib was initially administered once daily at 37.5 mg per day on days 1-14 of a 21-day cycle, in which irinotecan 250 mg m(-2) was given on day 1. In a second cohort, the sunitinib dose was reduced to 25 mg per day. Blood samples were collected for pharmacokinetic studies., Results: In the sunitinib 37.5 mg per day cohort, 3 out of 10 evaluable patients had objective responses, but dose-limiting toxicities (DLTs) of neutropenia, pneumococcal sepsis, and fatigue were observed. There were no DLTs in the sunitinib 25 mg per day cohort. Paired observations of pharmacokinetic parameter values of sunitinib and irinotecan alone vs the combination did not reveal significant drug-drug interactions. The maximum tolerated dose was defined as sunitinib 25 mg per day (days 1-14) with irinotecan 250 mg m(-2) (day 1), but no activity was observed at this dose., Conclusion: Although a higher sunitinib dose of 37.5 mg per day (days 1-14) with irinotecan showed preliminary evidence of antitumour activity, this dose was poorly tolerated. Therefore, this particular combination will not be pursued for further studies.

Background: Because sunitinib can induce extensive necrosis in metastatic renal cell cancer (mRCC), we examined whether criteria defined by Choi might be valuable to predict early sunitinib efficacy., Methods: Computed tomography was used for measurement of tumour lesions in mm and lesion attenuation in Hounsfield units (HUs). According to Choi criteria partial response (PR) was defined as > or =10% decrease in size or > or =15% decrease in attenuation., Results: A total of 55 mRCC patients treated with sunitinib were included. At first evaluation, according to the Response Evaluation Criteria in Solid Tumours (RECIST) 7 patients had PR, 38 stable disease (SD), and 10 progressive disease (PD), whereas according to Choi criteria 36 patients had PR, 6 SD and 13 PD. Median tumour attenuation decreased from 66 to 47 HUs (P< or =0.001). In patients with PR, Choi criteria had a significantly better predictive value for progression-free survival and overall survival (both Ps<0.001) than RECIST (P=0.685 and 0.191 respectively). The predictive value for RECIST increased (P=0.001 and <0.001 respectively), when best response during treatment was taken into account., Conclusion: Choi criteria could be helpful to define early mRCC patients who benefit from sunitinib, but the use of these criteria will not change the management of these patients.

Sunitinib has been registered for the treatment of advanced renal cell cancer (RCC). As patient inclusion was highly selective in previous studies, experience with sunitinib in general oncological practice remains to be reported. We determined the efficacy and safety of sunitinib in patients with advanced RCC included in an expanded access programme. ECOG performance status >1, histology other than clear cell and presence of brain metastases were no exclusion criteria. Eighty-two patients were treated: 23% reached a partial response, 50% had stable disease, 20% progressed and six patients were not evaluable. Median progression-free survival (PFS) was 9 months and median overall survival (OS) was 15 months. Importantly, 47 patients (57%) needed a dose reduction, 35 (43%) because of treatment-related adverse events, 10 (12%) because of continuous dosing, and two because of both. Stomatitis, fatigue, hand-foot syndrome and a combination of grade 1-2 adverse events were the most frequent reasons for dose reduction. In 40 patients (49%), there was severe toxicity, defined as dose reduction or permanent discontinuation, which was highly correlated with low body surface area, high age and female gender. On the basis of age and gender, a model was developed that could predict the probability of severe toxicity.

CD13/Aminopeptidase N (CD13) is known to play an important role in tumour cell invasion. We examined whether basic fibroblast growth factor (bFGF) is involved in the regulation of CD13 expression in human melanoma cells. 1F6 human melanoma cells were stably transfected with constructs encoding either the 18 kDa (18 kD) or all (ALL) bFGF isoform proteins. We observed highly increased CD13 mRNA and protein expression in the 1F6 clones regardless of the overexpression of either the 18 kD or all isoform proteins. Neutral aminopeptidase activity was increased five-fold and could be inhibited by bestatin and the CD13-neutralising antibody WM15. The enhanced invasion through Matrigel, but not migration in a wound assay, was efficiently abrogated by both bestatin and WM15. Upregulation of CD13 expression was the result of increased epithelial and myeloid promoter activity up to 4.5-fold in 1F6-18 kD and 1F6-ALL clones. Interestingly, in a panel of human melanoma cell lines, a significant correlation (r(2)=0.883, P<0.05) between bFGF and CD13 mRNA and protein expression was detected. High bFGF and CD13 expression were clearly related with an aggressive phenotype. Taken together, our data indicate that high bFGF expression upregulates CD13 expression in human melanoma cells by activating both the myeloid and the epithelial CD13 promoter. In addition, we show that high bFGF and CD13 expression results in enhanced invasive capacity and metastatic behaviour of human melanoma cells.

BNP7787 (disodium 2,2'-dithio-bis-ethane sulphonate; Tavocept) is a novel agent developed to protect against cisplatin (cis-diammine-dichloroplatinum(II))-associated chronic toxicities. In this study, we determined the recommended dose of BNP7787 when preceding a fixed dose of cisplatin, the pharmacokinetics (PKs) and the possible reduction of saline hydration. Patients with advanced solid tumours received BNP7787 in escalating doses of 4.1-41 g m(-2) as a 15-min intravenous (i.v.) infusion followed by cisplatin 75 mg m(-2) as a 60-min i.v. infusion together with pre- and postcisplatin saline hydration in a volume of 2200 ml; cycles were repeated every 3 weeks. PK was carried out using BNP7787, cisplatin and the combination. Twenty-five patients were enrolled in stage I of the study to determine the recommended dose of BNP7787. No dose-limiting toxicity was reached. The highest dose level of 41 g m(-2) resulted in a low incidence of grade 2 toxicities, being nausea and vomiting, dry mouth or bad taste and i.v. injection site discomfort. Doses of BNP7787 > or = 18.4 g m(-2) did not show a drug interaction between BNP7787 and cisplatin. In stage II of the study, patients received a fixed dose of BNP7787 of 18.4 g m(-2) preceding cisplatin and were entered in prespecified reduced saline hydration steps. A total of 21 patients in cohorts of six to nine patients received reduced saline hydration of 1600 ml (step A), 1000 ml (step B) and 500 ml (step C). In step C, two out of six evaluable patients experienced grade 1 nephrotoxicity. Cisplatin acute toxicities in all 46 patients were as expected. Only five patients complained of paresthesias grade 1 and six developed slight audiometric changes. Partial tumour response was observed in four patients and stable disease in 15 patients. In conclusion, BNP7787 was tolerated well up to doses of 41 g m(-2). The recommended dose of 18.4 g m(-2) enabled safe reduction of the saline hydration schedule for cisplatin to 1000 ml. Further studies will assess whether BNP7787 offers protection against platinum-related late side effects.

In preclinical studies, BNP7787 (disodium 2,2'-dithio-bis-ethane sulphonate), the disulphide form of mesna, has demonstrated selective protection against cisplatin-induced nephrotoxicity due to conversion into mesna inactivating toxic platinum species. Mesna (sodium 2-mercapto ethane sulphonate), however, can affect the antitumour activity of cisplatin, while BNP7787 does not interfere with the antitumour activity. To understand the difference in interference with cisplatin-induced antitumour activity between BNP7787 and mesna as well to characterise the selective nephroprotection by BNP7787, the pharmacokinetics of BNP7787 and mesna, each given i.v. 1000 mg x kg(-1), were determined in plasma, kidney, liver, red blood cells (RBC), skeletal muscle and tumour of Fischer rats bearing subcutaneously implanted WARD colon tumours. The following results were obtained: (1). high concentrations of BNP7787 and mesna were observed in the plasma and kidney after administration of BNP7787 or mesna, except for mesna in plasma after BNP7787 administration; (2). in all other sampled compartments, the AUC values of both compounds were at least 5.5-fold lower than the corresponding values in kidney; (3). the AUC of mesna in plasma after mesna administration was comparable to the AUC of mesna in kidney after a dose of BNP7787 that can completely prevent cisplatin-induced nephrotoxicity in rats; (4). the AUC of mesna in plasma was five-fold higher relative to the AUC of mesna following BNP7787 administration (P<0.01). In conclusion, the five-fold higher AUC of mesna in plasma after mesna administration and the fact that mesna is more reactive with (hydrated) cisplatin than its disulphide form BNP7787 represent a plausible explanation as to why mesna administration can reduce the antitumour activity of cisplatin. After BNP7787 administration, the distribution of BNP7787 and mesna was restricted to the kidney, which confirmed the selective protection of the kidney by BNP7787.

DX-8951f (exatecan mesylate), a new water-soluble derivative of camptothecin, is currently being evaluated in phase II clinical trials. Resistance may be acquired when treating cancer patients with DX-8951f. Therefore, we selected a subline of the human ovarian cancer cell line A2780 for resistance against DX-8951f to investigate possible mechanisms of resistance. This DX-8951f-resistant subline, designated 2780DX8 (resistance factor=9.3), displayed a typical cross-resistance pattern including compounds, such as topotecan (resistance factor =34), SN-38 (resistance factor =47), mitoxantrone (resistance factor =59) and doxorubicin (resistance factor =2.9), which have previously been associated with the expression of breast cancer resistance protein. 2780DX8 cells did not show changes in the topoisomerase I gene, in topoisomerase I protein levels or catalytic activity. Overexpression of breast cancer resistance protein could be detected, both at the mRNA and protein level, while staining for Pgp, MRP1, or LRP was negative. GF120918, an inhibitor of breast cancer resistance protein, was able to reverse the DX-8951f-induced resistance in 2780DX8 cells. In vivo experiments in well-established 2780DX8 human tumour xenografts demonstrated that the growth inhibition induced by CPT-11 was more affected by breast cancer resistance protein expression than that of DX-8951f. These data indicate for the first time that DX-8951f is able to induce breast cancer resistance protein as a mechanism of resistance. Breast cancer resistance protein, however, results in only minor reduction of antitumour activity of DX-8951f which is an advantage over topotecan and CPT-11/SN-38.

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug., (Copyright 2002 Cancer Research UK)

The doxorubicin (DOX) prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-beta-glucuronyl carbamate (DOX-GA3) was synthesised for specific activation by human beta-glucuronidase, which is released in necrotic areas of tumour lesions. This novel prodrug was completely activated to the parent drug by human beta-glucuronidase with V(max)= 25.0 micromol x min(-1) x mg(-1) and K(m) = 1100 microM. The pharmacokinetics and distribution of DOX-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3) were determined and compared with DOX. Administration of DOX at 8 mg x kg(-1) i.v. (maximum tolerated dose, MTD) to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 16.4 microM (t = 1 min). A 7.6-times lower peak plasma concentration of DOX was measured after injection of DOX-GA3 at 250 mg x kg(-1) i.v. (50% of MTD). In normal tissues the prodrug showed peak DOX concentrations that were up to 5-fold (heart) lower than those found after DOX administration. DOX-GA3 activation by beta-glucuronidase in the tumour yielded an almost 5-fold higher DOX peak concentration of 9.57 nmol x g(-1) (P< 0.05) than the peak concentration of only 2.14 nmol x g(-1) observed after DOX. As a consequence, the area under the curve of DOX calculated in tumour tissue after DOX-GA3 (13.1 micromol x min(-1) x g(-1)) was 10-fold higher than after DOX (1.31 micromol x min(-1) x g(-1)). The anti-tumour effects of DOX-GA3 and DOX were compared at equitoxic doses in OVCAR-3 xenografts at a mean tumour size of 125 mm(3). The prodrug given i.v. at 500 mg x kg(-1) weekly x 2 resulted in a maximum tumour growth inhibition of 87%, while the standard treatment with DOX at a dose of 8 mg x kg(-1) i.v. weekly x 2 resulted in a maximum tumour growth inhibition of only 56%. Treatment with DOX-GA3 was also given to mice with larger tumours containing more necrosis. For tumours with a mean size of 400 mm(3) the specific growth delay by DOX-GA3 increased from 2.7 to 3.9. Our data indicate that DOX-GA3 is more effective than DOX and suggest that the prodrug will be specifically advantageous for treatment of advanced disease., (Copyright 2001 Cancer Research Campaign.)

Multidrug resistance (MDR) and more specifically the expression of P-glycoprotein (Pgp) have been studied extensively in vitro. Unfortunately, it appears that the predictive value of MDR recognized in vitro is mostly an incorrect measure to determine the responsiveness of a particular tumour in the clinic. This misunderstood or overvalued role of MDR might explain the failure of strategies to reverse Pgp function by the use of modulators in solid tumours. To obtain more insight in in vivo drug resistance we investigated a panel of 15 human ovarian cancer xenografts consisting of the most common histological subtypes known in ovarian cancer patients. The response rate to cisplatin, cyclophosphamide and doxorubicin in the xenografts resembled the results of phase II trials with these agents in ovarian cancer patients. This resemblance justifies drug resistance studies in this experimental in vivo human tumour system. We determined the expression levels of MDR 1, MRP 1, LRP and topoisomerase IIalpha mRNA by the RNase protection assay and the presence of MRP1 and LRP proteins by immunohistochemistry. The S-phase fraction was investigated as a separate parameter by flow cytometry. In none of the 15 ovarian cancer xenografts was MDR 1 expression detectable. The expression levels of MRP 1 and LRP were low to moderate and resembled the presence of the MRP1 and LRP proteins. There was a weak, inverse relationship between the expression levels of LRP and sensitivity to cisplatin and cyclophosphamide (r = -0.44 and -0.45), but not to doxorubicin. The levels of topoisomerase IIalpha varied among the xenografts (0.73-2.66) and failed to correlate with doxorubicin resistance (r = 0.14). The S-phase fraction, however, showed a relation with the sensitivity to cisplatin (r = 0.66). Among the determinants studied in ovarian cancer in vivo, LRP mRNA and the S-phase fraction were the best predictive factors for drug response and most specifically for the activity of cisplatin., (Copyright 2000 Cancer Research Campaign.)

We have established ten transplantable human soft-tissue sarcoma (STS) xenografts grown as subcutaneous tumours in the nude mouse. Nine xenografts originated from patients that needed chemotherapy in the course of their disease. The xenografts were tested for their sensitivity to maximum tolerated doses of five anti-cancer agents. Growth of treated tumours was expressed as a percentage of control tumour growth and a growth inhibition > 75% was measured for doxorubicin in 20% of the STS xenografts, for cyclophosphamide in 30%, for ifosfamide in 20%, for vincristine in 20%, whereas etoposide was not effective in the STS xenografts. In three out of ten STS xenografts MDR1 mRNA was detectable, but this was not related to the resistance against doxorubicin, vincristine or etoposide. Topoisomerase IIalpha mRNA expression levels did not reflect sensitivity to doxorubicin or etoposide. In all STS tissues, however, these levels were lower than topoisomerase IIalpha mRNA in a drug-sensitive human ovarian cancer xenograft. Glutathione concentrations and the activities of glutathione S-transferase, glutathione peroxidase and glutathione reductase were not related to resistance against the alkylating agents or doxorubicin. Of interest, in all STS tissues, glutathione S-transferase pi was the predominant isoenzyme present. In conclusion, chemosensitivity of the STS xenografts reflects clinical response rates in phase II trials on the same compounds in adult STS patients. Relatively low levels of topoisomerase IIalpha mRNA may partly account for intrinsic resistance against, for example, doxorubicin. Additional factors must contribute to moderate responsiveness to alkylating agents.

The prodrug N-[4-(daunorubicin-N-carbonyl-oxymethyl)phenyl] O-beta-glucuronyl carbamate (DNR-GA3) was synthesized for specific activation by human beta-glucuronidase, released in necrotic areas of tumour lesions. In vitro, DNR-GA3 was 18 times less toxic than daunorubicin (DNR) and the prodrug was completely activated to the parent drug by human beta-glucuronidase. The maximum tolerated dose of DNR-GA3 in nude mice bearing s.c. human ovarian cancer xenografts was 6-10 times higher than that of DNR. The prodrug was cleared more rapidly from the circulation (elimination t1/2 = 20 min) than the parent drug (elimination t1/2 = 720 min). The anti-tumour effects of DNR-GA3 and DNR were investigated in four different human ovarian cancer xenografts OVCAR-3, FMa, A2780 and MRI-H-207 at a mean tumour size between 100 and 200 mm3. In three out of four of these tumour lines, the prodrug given i.v. at the maximum tolerated dose ranging from 150 to 250 mg kg(-1) resulted in a maximum tumour growth inhibition from 82% to 95%. The standard treatment with DNR at a dose of 8 mg kg(-1) given i.v. weekly x 2 resulted only in a maximum tumour growth inhibition from 40% to 47%. Tumour line FMa did not respond to DNR, nor to DNR-GA3. Treatment with DNR-GA3 was also given to mice with larger tumours that would contain more necrosis (mean size 300-950 mm3). The specific growth delay by DNR-GA3 was extended from 2.1 to 4.4 in OVCAR-3 xenografts and from 4.4 to 6.0 in MRI-H-207 xenografts. Our data indicate that DNR-GA3 is more effective than DNR and may be especially of use for treatment of tumours with areas of necrosis.

It has been recognized that the tumour markers alpha-fetoprotein (AFP) and human chorionic gonadotrophin (HCG) may show a transient elevation after the initiation of chemotherapy in non-seminomatous testicular cancer. We investigated the prognostic importance of these so-called marker surges in a cohort of patients treated with cisplatin combination chemotherapy between 1983 and 1991. A total of 669 patients were studied. Of 352 patients who had an elevated AFP at the start of treatment and for whom we had data at both day 1 and day 8, 101 (29%) had a surge. Of 317 patients for whom we had data for HCG, 80 patients (25%) had a surge. It was found that an AFP surge was a strong adverse prognostic factor for progression [hazard ratio (HR) 2.28, P=0.005]. There was no statistically significant difference in survival (HR 1.65, P=0.13). There was no prognostic significance of a HCG surge, either for progression or for survival. To investigate whether a surge was an independent prognostic factor for progression and survival, multivariate Cox regression models were fitted using the independent prognostic factors for progression and survival and the surge/decline variable. An AFP surge was retained in the final model for progression. A HCG surge was of no prognostic importance for progression or survival. We conclude that an AFP surge has an adverse prognostic significance, independent of pretreatment characteristics.

Irinotecan (CPT-11) is a semisynthetic camptothecin derivative with a broad spectrum of anti-tumour activity. Carboxylesterase (CE) catalyses the conversion of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin), the active form of CPT-11. The antiproliferative effects of CPT-11 and SN-38, CE-activity and topoisomerase I protein expression were investigated in five human small-cell lung cancer (SCLC) cell lines and four human non-small-cell lung cancer (NSCLC) cell lines. Antiproliferative activity, expressed as IC50 values, was determined using the MTT assay. CPT-11 was significantly more active in SCLC than in NSCLC cell lines (P = 0.0036), whereas no significant difference between histological types was observed with SN-38. A significant correlation (r2 = 0.52, P = 0.028) was observed between CE activity and chemosensitivity to CPT-11 but not to SN-38, and significantly higher CE activity was observed in SCLC compared with NSCLC cell lines (P = 0.025). Western blotting experiments showed topoisomerase I protein expressions within a factor of 2, and a granular nuclear staining was detectable in all cell lines by immunocytochemistry of cytospins. No correlation was observed between protein expression and sensitivity to CPT-11 or SN-38. Cellular and medium concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography (HPLC) in one SCLC cell line with high CE activity and high sensitivity to CPT-11, and one NSCLC cell line with low sensitivity to CPT-11 and CE activity. Intracellular concentrations of CPT-11 and SN-38 were higher in the SCLC cell line, and this was associated with an increase in cellular uptake of CPT-11 compared with the medium, and an increased intracellular formation of SN-38. In conclusion, CE activity appears to be associated with higher sensitivity to CPT-11 in human lung cancer cell lines and may partly explain the difference in the in vitro sensitivity to CPT-11 between SCLC and NSCLC cells. The assessment of CE activity in clinical material of lung cancer patients undergoing treatment with CPT-11 may be warranted. However, other mechanisms may influence sensitivity to CPT-11, possibly including drug transport.

The relevance of P170-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) for the sensitivity to CPT-11 was investigated in human malignant cell lines as well as in human tumour xenografts. In vitro, the P-gp-positive sublines BRO/mdr1.1 (transfected with MDR1) and 2780AD were slightly cross-resistant against carboxylesterase-activated CPT-11. Cross-resistance against SN-38 was present in 2780AD cells, but not in BRO/mdr1.1 cells. The P-gp modulators BIBW22BS, verapamil and dexniguldipine partly reversed the resistance against CPT-11 in the P-gp-positive sublines. BIBW22BS was the most effective modulator in the reversal of the resistance against carboxylesterase-activated CPT-11 as well as against SN-38 in the 2780AD subline. In contrast to doxorubicin and vincristine, the BRO/mdr1.1 xenografts were at least as sensitive to CPT-11 as the BRO xenografts. The 2780AD xenografts were slightly less sensitive than the parent tumours, but there was no difference in topoisomerase I DNA unwinding activity. Therefore, the high retention of the multidrug-resistant phenotype of 2780AD cells in vivo may be the cause of the low cross-resistance against CPT-11. The MRP-positive subline GLC4/ADR was cross-resistant against carboxylesterase-activated CPT-11 and SN-38. GLC4/ADR cells, however, demonstrated a twofold lower topoisomerase I activity than GLC4 cells. Cross-resistance against the camptothecin derivatives was not apparent in the MRP-transfected subline of SW1573/S1. In conclusion, P-gp-positive cells show a low cross-resistance against CPT-11/SN38, which is only apparent with high P-gp expression in vivo. MRP does not seem to play a role in the sensitivity to CPT-11.

We have previously reported that amifostine potentiates the anti-tumour activity of carboplatin in mice. The present study was carried out in well-established human ovarian cancer xenografts OVCAR-3, A2780 and FMa grown subcutaneously in the nude mouse. It was found that a single dose of amifostine resulted in a higher increase in the anti-tumour activity of carboplatin than three doses of amifostine. A single dose of amifostine increased the AUC (area under the curve) values of total platinum in plasma ultrafiltrate (30.1 vs 18.2 microM x h), liver (307.7 vs 236.4 nmol g(-1) x h), kidney (500.8 vs 368.3 nmol g(-1) x h) and OVCAR-3 tumour tissue (184.0 vs 146.8 nmol g(-1) x h). Despite this increase in total platinum, a decrease in platinum (Pt)-DNA adduct levels was observed in liver, kidney and bone marrow, which was significant in liver. In tumour tissue an insignificant increase in Pt-DNA adduct levels, specifically the Pt-GG adduct, was observed after treatment with a single dose of amifostine, which may explain the increase in anti-tumour activity. The increase in the AUC of total platinum was probably caused by a reduction in body temperature, which was most severe after three doses of amifostine. The extreme hypothermia may be the reason that three doses of amifostine resulted in less potentiation of the efficacy of carboplatin.

We investigated the prognostic value of the serum half-life of human chorionic gonadotrophin (HCG) and alpha-fetoprotein (AFP) during induction chemotherapy and the relative prognostic importance of initial marker concentrations and marker half-life. Marker half-lives were calculated using two abnormal values observed between day 8 and day 22 of the first chemotherapy cycle. Moreover, analyses were carried out using day 43 as the second measurement point. Treatment failure at any time was chosen as the end point. The relative prognostic influence of marker half-lives and initial marker concentrations was tested in univariate and multivariate analyses. Half-lives were considered to be prolonged if > 3 days for HCG and > 6 days for AFP. In addition, we separated patients into those with half-lives > 6 days for HCG and those with half-lives > 10 days for AFP to examine whether these long half-lives were associated with a poor prognosis. A group of 669 patients treated with cisplatin combination chemotherapy was studied. Forty-two per cent of the patients had normal HCG and 37% had normal AFP at the start of chemotherapy. At day 22, HCG was still elevated in 138 patients and AFP in 211. At day 43, the numbers of these patients were 35 and 80 respectively. Based on the measurements obtained on day 8 and day 22, a half-life of HCG > 3 days or > 6 days and/or a half-life AFP > 6 days or > 10 days did not accurately predict treatment failure (P=0.413 and P=0.851, respectively; values obtained using tests for trend). However, initial marker concentrations of HCG and/or AFP > 1000 IU l(-1) were highly significant prognosticators for treatment failure (P=0.001 and P < 0.001 respectively), independent of half-life values. Half-lives calculated with the values obtained on day 43 did not contribute to the accuracy of the prediction of treatment failure. We conclude that half-lives of HCG and AFP during induction chemotherapy are inaccurate parameters for the prediction of treatment failure. In contrast, initial serum concentrations of HCG and AFP are highly significant in the prediction of unfavourable treatment outcome.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a haematopoietic growth factor with a wide variety of applications in the clinic. In early phase I studies the continuous intravenous (c.i.) route of administration was often used. Later it was shown that subcutaneous (s.c.) administration was also effective. The optimal route of administration remains, however, poorly defined, and no studies have made a direct comparison between these two routes of administration. We treated patients with advanced breast cancer with moderately high-dose doxorubicin and cylophosphamide and GM-CSF. The first 14 patients received GM-CSF by c.i, while subsequently 47 patients received it s.c. Comparison between the two groups showed that c.i. GM-CSF was more toxic in several respects. There was a higher need for erythrocyte and platelet transfusions and a significant deterioration in the performance status. This study indicates that subcutaneous GM-CSF is the preferred route of administration. Randomised trials are, however, needed to confirm these conclusions.

The low-affinity monoclonal antibody (MAb) chimeric 17-1A(c-17-1A) and the high-affinity MAb mouse 323/A3 (m-323/A3) were used to study the effect of the MAb affinity on the tumour uptake and efficacy of radioimmunotherapy in nude mice bearing subcutaneously the human ovarian cancer xenografts FMa, OVCAR-3 and Ov.Pe. Both MAbs are directed against the same pancarcinoma glycoprotein. In vitro, the number of binding sites on tumour cells at 4 degrees C was similar for both MAbs, but m-323/A3 had an approximately 5-fold higher affinity (1.3-3.0x10(9) M-1) than c-17-1A (3.0-5.4x10(8) M-1). This difference in affinity was more extreme at 37 degrees C, when no binding of c-17-1A could be observed. MAb m-323/A3 completely blocked binding of c-17-1A to tumour cells, whereas the reverse was not observed. Immunohistochemistry showed a similar but more intense staining pattern of m-323/A3 in human ovarian cancer xenografts than of c-17-1A. In vivo, the blood clearance in non-tumour-bearing nude mice was similar for both MAbs with terminal half-lives of 71.4 h for m-323/A3 and 62.7 h for c-17-1A. MAb m-323/A3 targeted better to tumour tissue, but was more heterogeneously distributed than c-17-1A. The cumulative absorbed radiation dose delivered by m-323/A3 to tumour tissue was 2.5- to 4.7-fold higher than that delivered by c-17-1A. When mice were treated with equivalent radiation doses of 131(I)m-323/A3 and 131(I)c-17-1A, based on a correction for the immunoreactivity of the radiolabelled MAbs, m-323/A3 induced a better growth inhibition in two of the three xenografts. When the radiation doses were adjusted to obtain a similar amount of radiation in the tumour c-17-1A was more effective in tumour growth inhibition in all three xenografts.

The therapeutic efficacy of gemcitabine, a new nucleoside analogue, was assessed in a variety of well-established human soft tissue sarcoma and ovarian cancer xenografts grown s.c. in nude mice. Tumour lines selected had different histological subtypes, growth rates and sensitivities to conventional cytostatic agents. The three different doses and schedules designed on the basis of a mean weight loss between 5% and 15% were i.p. injections of daily 3.5 mg kg-1 x 4, every 3 days 120 mg kg-1 x 4, and weekly 240 mg kg-1 x 2, which ultimately resulted in 19%, 10% and 4% toxic deaths, respectively. The weekly schedule induced > or = 50% growth inhibition in 2/4 soft tissue sarcoma and 4/6 ovarian cancer lines, while in three ovarian cancer lines > or = 75% growth inhibition was obtained. The anti-tumour effects of gemcitabine appeared to be similar or even better than previous data with conventional drugs tested in the same tumour lines. In comparison with the every 3 days schedule, the weekly and the daily schedule were less effective in 5/7 and 3/3 tumour lines (P < 0.001), respectively. In another experiment in three human tumour lines selected for their differential sensitivity to gemcitabine, weekly injections of 240 mg kg-1 x 6 did not result in a significant increase in the percentages of growth inhibition when compared to lower doses of 120 mg kg-1 or 60 mg kg-1 in the same schedule. However, the 240 mg kg-1 weekly x 6 schedule showed superior effects in 2/3 tumour lines in comparison with the same dose given every 2 weeks x 3 (P < 0.05). The preclinical activity of gemcitabine suggests that the drug can induce responses in soft tissue sarcoma and ovarian cancer patients. Our results further indicate that clinical trials of gemcitabine in solid tumour types should be designed on the basis of a schedule rather than a dose dependence.

N-l-leucyl-doxorubicin and vinblastine-isoleucinate can be considered as relatively non-toxic prodrugs from doxorubicin and vinblastine, respectively. A comparative analysis was carried out of the anti-tumour activity of the four compounds as well as vintriptol in four human ovarian cancer xenografts different in histology, growth rate and chemosensitivity. Injections were given i.v. weekly twice into mice bearing well-established s.c. tumours. At equitoxic doses, the amount of drug administered for N-l-leucyl-doxorubicin and vinblastine-isoleucinate was respectively 3-fold and 2-fold higher than the doses of the parent compound. N-l-leucyl-doxorubicin induced a growth inhibition > 50% in three out of four human ovarian cancer lines. The anti-tumour effects obtained were significantly better (P < 0.01) than in the case of doxorubicin. Vinblastine-isoleucinate studied in two of these lines could induce a growth inhibition of > 50%. This prodrug appeared slightly less effective than vinblastine. Insignificant growth inhibition (< 50%) was obtained by vintriptol.

The anti-pan carcinoma monoclonal antibody (MAb) 323/A3, linked to E. coli-derived beta-glucuronidase (GUS) was used to study the tumour-site-selective activation of the prodrug Epirubicin-glucuronide (Epi-glu). Epi-glu was isolated from the urine of patients treated with Epirubicin (Epi) by reversed phase chromatography on a silica-C18 column. Epi-glu was stable in human blood and was not converted into Epi by A2780, MCF-7, or OVCAR-3 cancer cells, despite the presence of intracellular GUS. The stability of the prodrug was confirmed in BALB/c mice. MAb 323/A3 and GUS were linked through a stable thioether bond. The conjugate (1:1) was purified by ion exchange and gel filtration chromatography. Binding to target cells revealed an immunoreactivity of at least 60% and good retention of enzyme activity. A protein dye (sulforhodamine B) assay was used to analyse cytotoxicity. Epi (IC50 of 0.003-0.2 microM) was 100-1,000 times more toxic than Epi-glu (IC50 of greater than 20 microM), when cancer cells were exposed for 4 or 24 h to the drugs. The low cytotoxicity of Epi-glu was most likely due to the reduced cellular uptake rate of the prodrug (2.7 pmol 10(-6) cells min-1) as compared to that of the parent compound (25 pmol 10(-6) cells min-1). Pretreatment of antigen-positive cells with the 323/A3-GUS conjugate prior to prodrug exposure completely restored cytotoxicity as a result from hydrolysis of Epi-glu into Epi. Our results demonstrate that the 323/A3-GUS conjugate can specifically activate the stable non-toxic prodrug Epi-glu at the tumour cell level.

Monoclonal antibody (MAb) 139H2 was previously shown to localise specifically into ovarian cancer xenografts in nude mice. MAb 139H2 was compared with MAbs OC125 and OV-TL 3, all reactive with ovarian carcinomas, for the binding characteristics as IgG and F(ab')2 fragments with the use of the OVCAR-3 cell line grown in vitro and as s.c. xenografts. Immunoperoxidase staining of OVCAR-3 tissue sections with MAbs OC125 and 139H2 was heterogeneous, whereas MAb OV-TL 3 showed homogeneity. No differences in binding were observed between IgG and F(ab')2. The avidity expressed as apparent affinity constants of MAbs OC125, OV-TL 3 and 139H2 for OVAR-3 cells were 1 x 10(9) M-1, 1 x 10(9) M-1, and 1 x 10(8) M-1, while the number of antigenic determinants were 5 x 10(6), 1 x 10(6) and 7 x 10(6), respectively. In OVCAR-3 bearing nude mice the blood half-lives of the MAbs as IgG and F(ab')2 were approximately 50 h and 6 h, respectively. Maximum tumour uptake for the whole MAbs OC125, OV-TL 3, 139H2 and a control MAb 2C7 was 8.5%, 17.7%, 11.1% and 2.5% of the injected dose g-1, reached at 72 h after injection. For the respective F(ab')2 fragments, the maximum values were 5.2%, 10.0%, 5.5% and 1.9% of the injected dose g-1, reached between 6 h and 15 h. Tumour to non-tumour ratios were more favourable for the F(ab')2 fragments as compared to those for MAbs as IgG. Biodistribution in mice bearing a control tumour confirmed the specificity of tumour localisation of MAbs OC125, OV-TL 3 and 139H2. After injection of a tracer dose of 10 microCi of radiolabelled MAbs OC125, OV-TL 3 and 139H2 as IgG, tumours received 38 cGy, and 9 cGy. In our OVCAR-3 model, a ranking in efficiency in tumour localisation would indicate MAb OV-TL 3 as most favourable MAb, but cross-reactivity with subpopulations of human white blood cells might hamper its clinical use. Dosimetric data indicate a 4-fold higher radiation absorbed dose to tumours for IgG compared with F(ab')2 fragments.