Prognostic markers for colorectal cancer: estimating ploidy and stroma.

Background: We report here the prognostic value of ploidy and digital tumour-stromal morphometric analyses using material from 2624 patients with early stage colorectal cancer (CRC). Patients and methods: DNA content (ploidy) and stroma-tumour fraction were estimated using automated digital imaging systems and DNA was extracted from sections of formalin-fixed paraffin-embedded (FFPE) tissue for analysis of microsatellite instability. Samples were available from 1092 patients recruited to the QUASAR 2 trial and two large observational series (Gloucester, n = 954; Oslo University Hospital, n = 578). Resultant biomarkers were analysed for prognostic impact using 5-year cancer-specific survival (CSS) as the clinical end point. Results: Ploidy and stroma-tumour fraction were significantly prognostic in a multivariate model adjusted for age, adjuvant treatment, and pathological T-stage in stage II patients, and the combination of ploidy and stroma-tumour fraction was found to stratify these patients into three clinically useful groups; 5-year CSS 90% versus 83% versus 73% [hazard ratio (HR) = 1.77 (95% confidence interval (95% CI): 1.13-2.77) and HR = 2.95 (95% CI: 1.73-5.03), P < 0.001]. Conclusion: A novel biomarker, combining estimates of ploidy and stroma-tumour fraction, sampled from FFPE tissue, identifies stage II CRC patients with low, intermediate or high risk of CRC disease specific death, and can reliably stratify clinically relevant patient sub-populations with differential risks of tumour recurrence and may support choice of adjuvant therapy for these individuals.

Variation in the availability of cancer drug generics in the United States of America.

While most of the attention and spending in the oncology community in the United States has been focused on the remarkable scientific inventions of the newer targeted drugs, the shortage of the older essential cancer drugs that are off patent, mostly generics and injectables, has a threatening impact on the health of cancer patients, the execution of clinical trials and the identification of newer drugs and thus impacts upon the burden of costs and pressures on the health system in the United States. It is a part of the problem of the scarcity of generics across all medical specialties, but its oncology is particularly vulnerable. The problem in The United States has been increasing since the beginning of the 21st century until the 2011; since then there has been some improvement in 2012 and the first two quarters of 2013. In the second quarter of 2012, there were 211 active shortages, down from 246 reports of active shortages in the same quarter of 2011. The Food and Drug Administration (FDA) officials ascribe the improvement to efforts that the agency made after President Obama issued an executive order in 2012 that impel the FDA to obtain early reports from companies about potential shortages. The drivers of the shortages are multi-factorial. But are largely economic and are due to the lack of incentives to produce generics. There are efforts from the US government, politicians and the medical, pharmacy and oncology communities. However, the problem is still serious. There is a general agreement that efforts so far have not been adequate, and that there is a need for addressing effectively the fundamentals and the underlying causes. There is a lot that could be done in the United States and across the world to improve the accessibility of economically sustainable better value cancer drugs regardless of whether they are brand or generics and aiming at a win-win outcome for all stakeholders.

The proportion of tumor-stroma as a strong prognosticator for stage II and III colon cancer patients: validation in the VICTOR trial.

BACKGROUND: The intra-tumor stroma percentage in colon cancer (CC) patients has previously been reported by our group as a strong independent prognostic parameter. Patients with a high stroma percentage within the primary tumor have a poor prognosis. PATIENTS AND METHODS: Tissue samples from the most invasive part of the primary tumor of 710 patients (52% Stage II, 48% Stage III) participating in the VICTOR trial were analyzed for their tumor-stroma percentage. Stroma-high (>50%) and stroma-low (≤50%) groups were evaluated with respect to survival times. RESULTS: Overall and disease-free survival times (OS and DFS) were significantly lower in the stroma-high group (OS P<0.0001, hazard ratio (HR)=1.96; DFS P<0.0001, HR=2.15). The 5-year OS was 69.0% versus 83.4% and DFS 58.6% versus 77.3% for stroma-high versus stroma-low patients. CONCLUSION: This study confirms the intra-tumor stroma ratio as a prognostic factor. This parameter could be a valuable and low cost addition to the TNM status and next to current high-risk parameters such as microsatellite instability status used in routine pathology reporting. When adding the stroma-parameter to the ASCO criteria, the rate of 'undertreated' patients dropped from 5.9% to 4.3%, the 'overtreated' increased with 6.8% but the correctly classified increased with an additional 14%.

The TERT variant rs2736100 is associated with colorectal cancer risk.

BACKGROUND: Polymorphic variation at the 5p15.33 (TERT-CLPTM1L) locus is associated with the risk of many cancers but a relationship with colorectal cancer (CRC) risk has yet to be defined. METHODS: We used data from six genome-wide association studies (GWAS) of CRC, linkage disequilibrium mapping and imputation, to examine the relationship between 73 single-nucleotide polymorphisms at 5p15.33 and CRC risk in detail. RESULTS: rs2736100, which localises to intron 2 of TERT, provided the strongest evidence of an association with CRC (P=2.28 × 10⁻4). The association was also shown in an independent series of 10 047 CRC cases and 6918 controls (P=0.02). A meta-analysis of all seven studies (totalling 16 039 cases, 16 430 controls) provided increased evidence of association (P=2.49 × 10⁻5; per allele odds ratio=1.07). The association of rs2736100 on CRC risk was shown to be independent of 15 low-penetrance variants previously identified. CONCLUSION: The rs2736100 association demonstrates an influence of variation at 5p15.33 on CRC risk and further evidence that the 5p15.33 (TERT-CLPTM1L) locus has pleiotropic effects (reflecting generic or lineage-specific effects) on cancer risk.

A two-phase case-control study for colorectal cancer genetic susceptibility: candidate genes from chromosomal regions 9q22 and 3q22.

BACKGROUND: Colorectal cancer (CRC) is the second cause of cancer-related death in the Western world. Much of the CRC genetic risk remains unidentified and may be attributable to a large number of common, low-penetrance genetic variants. Genetic linkage studies in CRC families have reported additional association with regions 9q22-31, 3q21-24, 7q31, 11q, 14q and 22q. There are several plausible candidate genes for CRC susceptibility within the aforementioned linkage regions including PTCH1, XPA and TGFBR1 in 9q22-31, and EPHB1 and MRAS in 3q21-q24. METHODS: CRC cases and matched controls were from EPICOLON, a prospective, multicentre, nationwide Spanish initiative, composed of two independent phases. Phase 1 corresponded to 515 CRC cases and 515 controls, whereas phase 2 consisted of 901 CRC cases and 909 controls. Genotyping was performed for 172 single-nucleotide polymorphisms (SNPs) in 84 genes located within regions 9q22-31 and 3q21-q24. RESULTS: None of the 172 SNPs analysed in our study could be formally associated with CRC risk. However, rs1444601 (TOPBP1) and rs13088006 (CDV3) in region 3q22 showed interesting results and may have an effect on CRC risk. CONCLUSIONS: TOPBP1 and CDV3 genetic variants on region 3q22 may modulate CRC risk. Further validation and meta-analysis should be undertaken in larger CRC cohorts.

A phase I and pharmacokinetic study of lapatinib in combination with infusional 5-fluorouracil, leucovorin and irinotecan.

BACKGROUND: This study determined the optimally tolerated regimen (OTR) of oral lapatinib administered in combination with infusional 5-fluorouracil (5-FU), leucovorin and irinotecan (FOLFIRI) and assessed the safety, tolerability and pharmacokinetics of the combination. PATIENTS AND METHODS: Twenty-five patients were enrolled; 12 patients were treated at three dose levels to determine OTR; then 13 patients were treated at OTR to evaluate the pharmacokinetics of the combination. RESULTS: The 2-weekly OTR comprised lapatinib 1250 mg/day with irinotecan 108 mg/m(2) (day 1) and leucovorin 200 mg/m(2), 5-FU bolus 240 mg/m(2) and 5-FU infusion 360 mg/m(2) (days 1 and 2); doses of 5-FU and irinotecan represent a 40% reduction in dose compared to conventional FOLFIRI. Dose-limiting toxicities were grade 3 diarrhoea and grade 4 neutropenia. Co-administration of lapatinib increased the area under the plasma concentration-time curve of SN-38, the active metabolite of irinotecan, by an average of 41%; no other pharmacokinetic interactions were observed. Of 19 patients evaluable for disease response assessment, four patients had partial response and nine patients had stable disease. CONCLUSION: The combination of lapatinib and FOLFIRI is safe and demonstrates clinical activity; the documented PK interaction can effectively be compensated by lowering the doses of 5-FU and irinotecan. This regime may be further tested in a phase II trial.

Standard operating procedure for the collection of fresh frozen tissue samples.

Studies using fresh-frozen tissue samples originating from different centres, as is often the case in EORTC related translational research, can show conflicting research results due to heterogeneity in the quality of samples and associated data from each centre. The development of infrastructure for the European Human Frozen Tumour Tissue Bank (TuBaFrost) anticipated this problem and Standard Operating Procedures (SOPs) have been developed to ensure samples collected are of consistent high quality and variation in research results is minimised. The SOPs drew on the best practice standard workflows and operating procedures employed by members of the TuBaFrost Consortium and key tissue bank initiatives worldwide. It was essential to provide workable solutions that reflect the variety in infrastructure and resources at the potential collecting centres and also the fact that it is not necessary to standardise every step of the collection and storage process in order to collect high quality tissue. Hence, the TuBaFrost SOPs detail the compulsory measures that must be implemented in order to become a TuBaFrost collecting centre and also make advisory recommendations regarding the less critical factors. Accordingly, the TuBaFrost SOPs are very flexible and to illustrate this the complete SOP for collecting, freezing and storing tissue at the Erasmus MC Tissue Bank is included. These TuBaFrost SOPs could equally be applicable to centres collecting samples for EORTC related translational research studies in order to standardise sample quality and produce reliable and reproducible research results.

Preclinical and phase I studies with rhizoxin to apply a pharmacokinetically guided dose-escalation scheme.

BACKGROUND: Rhizoxin is a new macrocyclic lactone isolated from the fungus Rhizopus chinensis which displays broad-spectrum antitumor activity against murine and human tumor xenografts and has activity against a number of vincristine-resistant tumors in vitro and in vivo. PURPOSE: This study describes the preclinical and clinical pharmacology of rhizoxin to apply a pharmacokinetically guided dose-escalation (PGDE) strategy during the phase I trial. METHODS: Rhizoxin was administered by a single intravenous bolus injection to female BALB/c mice over the dose range 7.5-18 mg/m2 from which we derived the dose that was lethal to 10% and 50% of the mice (i.e., LD10 and LD50, respectively). The LD10 was 11.7 +/- 0.7 mg/m2 (mean +/- SD), and the LD50 was 14.7 +/- 0.6 mg/m2. Pharmacokinetic studies were integrated with the toxicity study in female BALB/c mice at one-tenth the LD10, one-half the LD10, and the LD10 (i.e., 1.2, 6, and 12 mg/m2, respectively). From these data, a target area under the plasma drug concentration versus time curve (AUC) (i.e., 40% of the LD10 AUC) was calculated for clinical studies. Phase I studies were initiated at 0.8 mg/m2 (one-tenth the equivalent LD10 in male CD1 mice), with the intent of escalating the dose by an extended factor-of-two method until the target AUC and/or maximum tolerated dose (MTD) was reached. RESULTS: The major drug toxic effects in mice were body weight loss, sluggishness, ataxia, transient changes in hematological parameters, and hematuria. Diarrhea was universal at doses greater than 9 mg/m2, and hind limb paralysis was observed in one of 10 mice, but only at supralethal doses (18 mg/m2). Rhizoxin pharmacokinetics were best described by a two-compartment open model (half-life [t 1/2] alpha = 4.4 minutes +/- 0.9 minute [mean +/- SD], and t 1/2 beta = 84 minutes +/- 20 minutes at 12 mg/m2) and found to be nonlinear with respect to dose. At doses of 1.2, 6, and 12 mg/m2, the respective AUC values were 1.3, 22.4, and 70.6 microM x minute. From these data, a target AUC value of 28 microM x minute (40% of the LD10 AUC) was derived. Rhizoxin was not detectable in patient plasma (less than 5 ng/mL at 0.8 and 1.6 mg/m2), and doses had to be escalated by conventional methods. Myelosuppression was dose limiting in patients: Seven of eight treated at 2.6 mg/m2 experienced World Health Organization grade 3-4 neutropenia, and five of eight developed mucositis. The AUC values at the human MTD (2.6 mg/m2) were in the range of 0.41-1.01 microM x minute, considerably lower than the target AUC of 28 microM x minute. CONCLUSION AND IMPLICATIONS: Although PGDE schemes have been successfully employed for other antitumor agents, this methodology could not be applied during the phase I trial of rhizoxin. PGDE studies in the future may incorporate comparative murine versus human metabolism studies in vitro with phenotyped liver microsomes. It may also be useful to assess the comparative myelotoxicity of a new drug by performing in vitro cytotoxicity studies on mouse and human bone marrow stem cells.

The prognostic significance of Bcl-2 and p53 expression in ovarian carcinoma.

Advanced ovarian cancer is characterized by poor prognosis and the development of resistance to chemotherapy. We have found that Bcl-2 and p53, two proteins implicated in the control of apoptosis, are differently expressed in the ovarian cell line A2780 and its cisplatin-resistant variant 2780CP, with the resistant line overexpressing both proteins. Transfection of the A2780 cells with a Bcl-2- or p53-expressing plasmid increases resistance to various drugs, including cisplatin, suggesting that Bcl-2 and p53 expression may influence the sensitivity of ovarian cancer cell lines to chemotherapy. Expression of these two proteins in vivo was determined by immunohistochemical staining of ovarian tumor biopsies from 70 patients. We found that Bcl-2 and p53 were expressed in 57 and 61% of specimens examined, respectively. Both p53 and Bcl-2 were found to be independent prognostic indicators of survival in ovarian cancer. Survival was poorer in patients with tumors expressing high levels of p53, whereas expression of Bcl-2 was associated with improved survival.

Phase I and pharmacokinetic study of flavone acetic acid.

Flavone acetic acid is the second in a series of compounds based on the flavonoid aglycone ring structure to be clinically evaluated in malignant disease. Preclinical studies have indicated that a minimum plasma level of 150 micrograms/ml is required before therapeutic efficacy (in a wide range of experimental tumors) is seen in mice; both in vitro and in vivo studies also suggest that the duration of drug exposure is crucial in determining activity. Thus a Phase I trial has been performed in a total of 54 patients using 3 schedules, i.e., a 1-, 3-, and 6-h infusion. In each case, treatment was given once weekly for a minimum of 3 weeks. The maximum tolerated doses were 6.4, 6.4, and 10.0 g/m2, respectively. Dose limiting toxicity was denoted by an intense feeling of warmth and flushing with a 1-h infusion, hypotension with a 3-h infusion, and hypotension and diarrhea with a 6-h infusion. No objective responses were seen in this Phase I trial. The recommended doses for Phase II trials of flavone acetic acid in Europe are 4.8 g/m2 over 1 h or 8.6 g/m2 over 6 h. At these doses the peak plasma concentrations obtained are 650 and 388 micrograms/ml, respectively. Total drug exposure (assessed by an area under the curve greater than 100 micrograms/ml) was approximately 50% greater for the 6-h schedule. This Phase I trial indicates that peak plasma concentrations associated with experimental activity are achievable in humans, although optimal drug exposure times have not yet been defined.

Phase I and pharmacokinetic study of LM985 (flavone acetic acid ester).

We have conducted a Phase I and initial clinical pharmacological evaluation of LM985, the first of a series of compounds based on the flavone ring structure to be considered for clinical trial in malignant disease. The drug was administered i.v. to 26 patients with advanced cancer on an every-21-day schedule. Patients were treated at 14 dosage levels ranging from 10 to 1500 mg/m2. Dose limiting toxicity was identified as acute reversible hypotension occurring during drug infusion; no leukopenia, alopecia, hepatic toxicity, or renal toxicity was observed, but at the higher dose range, mild sedation was apparent. Twenty patients had measurable disease and were evaluable for response. One patient with colorectal carcinoma had stable disease after three courses of LM985; however, no other responses were seen. Pharmacokinetic and in vitro drug degradation studies imply that the ester LM985 is hydrolyzed to LM975 (flavone acetic acid) rapidly in vivo. LM975 is active in a variety of animal tumor models, but it does not have the cardiovascular side effects seen with LM985 (hypotension and bradycardia) in pithed or anesthetized rats. We would recommend that LM975 be considered for clinical trial, because it seems likely that substantially higher doses of LM975 than of LM985 can be given without dose limiting cardiovascular toxicity.

Characterization of the major metabolites of flavone acetic acid and comparison of their disposition in humans and mice.

Flavone acetic acid represents a novel chemical structure currently undergoing clinical investigation. Broad spectrum activity has been observed in preclinical animal screens, but at doses close to toxic in mice. Phase I clinical trials have established that equivalent plasma drug levels can be achieved in humans, but to date Phase II trials have not demonstrated significant activity in a range of tumor types. Little is known about the drug's biotransformation, although metabolites have been implicated in proposed mechanisms of action. In this paper, we have purified the two major human metabolites present in urine (also the only two metabolites detected in plasma) and characterized their structure, chemical properties, activity, and pharmacokinetics. Metabolite 1 (M1) was a glucuronide conjugated to the 8-acetic acid grouping (Mr 456), was chemically labile, and showed a strong tendency to undergo chemical rearrangement at mildly alkaline pH. Metabolite 2 (M2) was also a glucuronide (Mr 456) but appeared to be an unusual isomer of M1. Both were noncytotoxic. In patients, biotransformation represented the predominant mechanism of drug clearance with as much as 80% of a low dose (0.5 g/m2) recovered in urine as M1 and M2 after only 6 h. At high dose (4.8 to 8.6 g/m2, 1- to 6-h infusion) the appearance of peak concentrations of metabolites in plasma and urine was delayed, apparently due to saturation of glucuronidation pathways. This resulted in an overall reduction in drug clearance by 3- to 4-fold. Mice cleared flavone acetic acid much more slowly than patients (289 ml/h/m2 after 600 mg/m2 i.p. versus 2.3 liters/h/m2 after 4.8 g/m2-1-h i.v. infusion) without producing M1 or M2. A different metabolite, exhibiting characteristics of a conjugate, was detected at low concentrations in plasma, tissues, and tumor. Extensive metabolism to inactive products followed by their rapid clearance may contribute to the lack of activity so far seen in humans.

Phase I and pharmacokinetic study of taxotere (RP 56976) administered as a 24-hour infusion.

N-Debenzoyl-N-tert-butoxycarbonyl-10-deacytyl taxol (Taxotere, RP 56976) is a semisynthetic analogue of taxol, prepared from a noncytotoxic precursor extracted from the needles of the European yew tree (Taxus baccata L.). It has a broad spectrum of antitumor activity against a variety of transplantable tumors in mice. In vitro cytotoxicity assays suggest that it is 2-5-fold more potent than taxol. In this phase I study Taxotere was administered by 24 h i.v. infusion at 3-week intervals. Thirty patients with solid tumors refractory to conventional therapy were treated; 70 courses of Taxotere were administered at doses ranging from 10 to 90 mg/m2. Grade 4 neutropenia and grade 3 mucositis were dose limiting but reversible at 90 mg/m2. The pattern and grade of toxicity at this dose were similar in 3 heavily pretreated patients compared with 7 patients who had received a maximum of one previous chemotherapy regimen. Alopecia occurred at 55 mg/m2 and above. Other mild toxicities included phlebitis, diarrhea, emesis, and sensory peripheral neuropathy, but these were neither dose-limiting nor clearly dose-related. One patient treated at 70 mg/m2 had an anaphylactoid reaction following the second dose of Taxotere. No cardiovascular toxicity was observed. No partial or complete responses were documented. Plasma concentrations of Taxotere were determined by high-performance liquid chromatography, and end-of-fusion levels at the maximum tolerated dose exceeded drug concentrations which are cytotoxic in vitro. The maximum tolerated dose for Taxotere administered as a 24-h infusion is 90 mg/m2.

The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and Bcl-2.

Modulation of apoptosis may influence resistance to chemotherapy and therefore affect the outcome of cancer treatment. Ovarian cancer, one of the most fatal malignancies in women, is often associated with drug resistance but the cellular pathways contributing to this effect remain obscure. We have found that Bcl-2 and p53, two proteins implicated in the control of apoptosis, are frequently expressed in fresh biopsies of primary ovarian carcinoma. Examination of Bcl-2 and p53 protein levels in pairs of cis-platin sensitive and resistant ovarian cell lines demonstrated that the resistant variants over-express Bcl-2 and/or p53, apparently due to progressive expansion of Bcl-2 and/or p53 positive subpopulations during the in vitro development of resistance. Exogenous expression of Bcl-2 or a temperature sensitive mutant p53 (ts p53) in the ovarian cell line A2780 resulted in protection from drug-induced apoptosis and a delay in drug-mediated S-phase arrest. Interestingly, p53 accumulation in response to DNA damage induced by different agents was significantly delayed and reduced in the Bcl-2 transfectants compared to the control A2780 line, suggesting that Bcl-2 may act upstream of the p53 pathway. Similarly, the induction of Bax mRNA and protein was also found to be delayed in the presence of Bcl-2. Overall, our data provide further evidence for cross-talk between Bcl-2, p53 and Bax and suggest that these genes are important determinants of drug-induced apoptosis thereby modulating resistance to chemotherapy.

CD40-induced growth inhibition in epithelial cells is mimicked by Epstein-Barr Virus-encoded LMP1: involvement of TRAF3 as a common mediator.

CD40, a member of the tumour necrosis factor receptor family, is expressed on the surface of B lymphocytes where its ligation provides a potent survival signal. CD40 is also expressed in basal epithelial cells and in a number of different carcinomas where its function remains unknown. We observed that contrary to the studies in normal B cells, CD40 ligation in carcinoma cell lines and in normal primary epithelial cells resulted in growth inhibition and enhanced susceptibility to apoptosis induced by anti-neoplastic drugs, TNF-alpha, Fas and ceramide. This effect was also observed in CD40-transfected Rat-1 fibroblasts. The expression of Bcl-2 did not affect growth inhibition induced by CD40 ligation in epithelial cells but the Epstein - Barr Virus-encoded latent membrane protein 1 (LMP1) blocked the effect. Whilst transient expression of LMP-1 resulted in the inhibition of epithelial cell growth, this effect was not observed with a LMP1 mutant lacking the binding domain for TRAF3, a protein which may mediate signal transduction by interacting with the cytoplasmic domains of both CD40 and LMP1. Transient expression of TRAF3 also inhibited epithelial cell growth, whilst expression of a dominant-negative TRAF3 partially blocked the inhibitory effect of CD40 ligation and of transient LMP1 expression. These results suggest that CD40 regulates epithelial cell growth in a manner mimicked by LMP1 and implicate TRAF3 as a common mediator in the transduction of the growth inhibitory signals generated via the CD40 and LMP1 pathways.

Inhibition of phospholipid signalling and proliferation of Swiss 3T3 cells by the wortmannin analogue demethoxyviridin.

Growth factors and certain oncogenes activate a range of phospholipid-mediated signal transduction pathways resulting in cell proliferation. Demethoxyviridin (DMV), a structural analogue of wortmannin and recently reported as a potent inhibitor of phosphoinositide-3-kinase, inhibited bombesin plus insulin-stimulated increase in cell number in Swiss 3T3 cells, a model of cell proliferation. The drug produced cytostatic effects at concentrations below 1 microM and cytotoxic effects at 10 microM. In intact Swiss 3T3 cells DMV inhibited insulin-stimulated PI 3- and 4-kinases and bombesin-stimulated phospholipases C, D and A2 in the nanomolar range. DMV also inhibited bombesin-stimulated tyrosine phosphorylation of a range of proteins at nM concentrations. This study shows that DMV inhibited multiple stimulated signalling pathways which lead to increased Swiss 3T3 cell proliferation. A stable analogue of DMV may have chemotherapeutic potential.

Induction of apoptosis by chemotherapeutic drugs: the role of FADD in activation of caspase-8 and synergy with death receptor ligands in ovarian carcinoma cells.

Although ovarian tumours initially respond to chemotherapy, they gradually acquire drug resistance. The aims of this study were to identify how chemotherapeutic drugs with diverse cellular targets activate apoptotic pathways and to investigate the mechanism by which exposure to a combination of drugs plus death receptor ligands can increase tumour cell kill. The results show that drugs with distinct cellular targets differentially up-regulate TRAIL and TNF as well CD95L, but do not require interaction of these ligands with their receptor partners to induce cell death. Factors that were critical in drug-induced apoptosis were activation of caspases, with caspase-8 being activated by diverse drugs in a FADD-independent manner. Certain drugs also demonstrated some dependence on FADD in the induction of cell death. Caspase-9 was activated more selectively by chemotherapeutic agents. Combining ligation of death receptors with exposure to drugs increased tumour cell kill in both drug resistant cell lines and primary ovarian carcinoma cells, even though these cells were not sensitive to death receptor ligation alone. CD95L was more consistent at combining with drugs than TRAIL or TNF. Investigation of the mechanism by which a combination of drugs plus CD95 ligation can increase cell death showed that caspase-8 was activated in cells exposed to a combination of cisplatin and anti-CD95, but not in cells exposed to either agent alone.

Phase I clinical and pharmacokinetic study of leucovorin and infusional hepatic arterial fluorouracil.

PURPOSE: A phase I and pharmacokinetic trial was performed between October 1993 and June 1994 to determine the maximum-tolerated dose of hepatic arterial infusion (HAI) of fluorouracil (5-FU) and intravenous (IV) leucovorin (folinic acid; FA) in patients with hepatic metastases from colorectal cancer. PATIENTS AND METHODS: Forty-three patients received 310 courses of HAI chemotherapy administered over 48 hours every 2 weeks. The regimen consisted of FA 200 mg/m2 by IV infusion over 2 hours, followed by a loading dose of 5-FU 400 mg/m2 by HAI over 15 minutes, followed by a 22-hour infusion of 5-FU at doses ranging from 0.8 to 1.84 g/m2, with identical chemotherapy on day 2. Pharmacokinetic studies were performed to determine peak and steady-state plasma concentrations (Css) of 5-FU. RESULTS: Severe diarrhea and cardiac and neurologic toxicity were dose-limiting at 1.84 g/m2. The recommended dose for the 22-hour component of the schedule was 1.6 g/m2 and was associated with tolerable toxicity. A Css of 2.2 +/- 0.8 mumol/L for 5-FU was achieved on the recommended schedule, which compares favorably with conventional IV 5-FU regimens. Among 30 patients assessable for response, there were four complete responses and seven partial responses, and 12 patients with stable disease and seven with progressive disease, reported after 3 months (ie, six cycles) of therapy. CONCLUSION: A regimen that combines 5-FU and FA has been identified for regional chemotherapy in patients with hepatic metastases from colorectal cancer. The systemic levels of 5-FU achieved are similar to the conventional IV de Gramont regimen using an identical schedule of 5-FU and FA, which implies that this chemotherapy has the best of both worlds, ie, a regional advantage in delivering high drug concentrations to the target organ with adequate systemic cover for extrahepatic micrometastases.