C. Barbara

Phase III Trial of Infusional Fluorouracil, Leucovorin, Oxaliplatin, and Irinotecan (FOLFOXIRI) Compared With Infusional Fluorouracil, Leucovorin, and Irinotecan (FOLFIRI) As First-Line Treatment for Metastatic Colorectal Cancer: The Gruppo Oncologico Nord Ovest

PURPOSE The Gruppo Oncologico Nord Ovest (GONO) conducted a phase III study comparing fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI [irinotecan 165 mg/m2 day 1, oxaliplatin 85 mg/m2 day 1, leucovorin 200 mg/m2 day 1, fluorouracil 3,200 mg/m2 48-hour continuous infusion starting on day 1, every 2 weeks]) with infusional fluorouracil, leucovorin, and irinotecan (FOLFIRI). METHODS Selection criteria included unresectable metastatic colorectal cancer, age 18 to 75 years, and no prior chemotherapy for advanced disease. The primary end point was response rate (RR). RESULTS A total of 244 patients were randomly assigned. An increase of grade 2 to 3 peripheral neurotoxicity (0% v 19%; P < .001), and grade 3 to 4 neutropenia (28% v 50%; P < .001) were observed in the FOLFOXIRI arm. The incidence of febrile neutropenia (3% v 5%) and grade 3 to 4 diarrhea (12% v 20%) were not significantly different. Responses, as assessed by investigators, were, for FOLFIRI and FOLFOXIRI, respectively, complete, 6% and 8%; and partial, 35% and 58%, (RR, 41% v 66%; P = .0002). RR confirmed by an external panel was 34% versus 60% (P < .0001). The R0 secondary resection rate of metastases was greater in the FOLFOXIRI arm (6% v 15%; P = .033, among all 244 patients; and 12% v 36%; P = .017 among patients with liver metastases only). Progression-free survival (PFS) and overall survival (OS) were both significantly improved in the FOLFOXIRI arm (median PFS, 6.9 v 9.8 months; hazard ratio [HR], 0.63; P = .0006; median OS, 16.7 v 22.6 months; HR, 0.70; P = .032). CONCLUSION The FOLFOXIRI regimen improves RR, PFS, and OS compared with FOLFIRI, with an increased, but manageable, toxicity in patients with metastatic colorectal cancer with favorable prognostic characteristics. Further studies of FOLFOXIRI in combination with targeted agents and in the neoadjuvant setting are warranted.

An improved HPLC method for therapeutic drug monitoring of daunorubicin, idarubicin, doxorubicin, epirubicin, and their 13-dihydro metabolites in human plasma.

A single high-performance liquid chromatography (HPLC) method, suitable for the analysis of daunorubicin, idarubicin, doxorubicin, epirubicin, and their 13-dihydro metabolites is validated in the present study. Preparation of plasma samples was performed by a first extraction of analytes with a chloroform/1-heptanol mixture (9:1) and reextraction with ortophosphoric acid 0.1 M. The chromatographic analysis was carried out by reversed-phase isocratic elution of anthracyclines with a Supelcosil LC-CN 5 mm column (25 cm x 4.6 mm internal diameter; Supelco) and detection was accomplished by spectrofluorimetry at excitation and emission wavelengths of 480 and 560 nm, respectively. All anthracyclines eluted within 15 minutes of injection and the method appeared to be specific, because the chromatographic assay did not show interferences at the retention time of analytes. The linearity, evaluated over a concentration range of 0.4-10,000 ng/mL, gave regression coefficients better than 0.999, with recoveries of doxorubicin-doxorubicinol and epirubicin-epirubicinol of 67%-109% and 61%-109% respectively, and 93%-109% for the other compounds. The limits of detection and quantification were 0.4 ng/mL in a 50-mL sample (40 pg/injection) for all anthracyclines tested. The method proved to be precise and accurate, as the within-day and between-day coefficients of variation were less than 10% and the accuracy of the assay was in the range of 91%-107%. Overall results indicate that it is feasible to analyze all the anthracyclines used in clinical practice and their major metabolites with a single optimized method, thereby simplifying their monitoring in chemotherapeutic regimens of cancer patients.

Continuation or reintroduction of bevacizumab beyond progression to first-line therapy in metastatic colorectal cancer: final results of the randomized BEBYP trial

BACKGROUND The combination of bevacizumab with fluorouracil-based chemotherapy is a standard first-line treatment option in metastatic colorectal cancer (mCRC). We studied the efficacy of continuing or reintroducing bevacizumab in combination with second-line chemotherapy after progression to bevacizumab-based first-line therapy. PATIENTS AND METHODS In this phase III study, patients with mCRC treated with fluoropyrimidine-based first-line chemotherapy plus bevacizumab were randomized to receive in second-line mFOLFOX-6 or FOLFIRI (depending on first-line regimen) with or without bevacizumab. The primary end point was progression-free survival. To detect a hazard ratio (HR) for progression of 0.70 with an α and β error of 0.05 and 0.20, respectively, 262 patients were required. RESULTS In consideration of the results of the ML18147 trial, the study was prematurely stopped. Between April 2008 and May 2012, a total of 185 patients were randomized. Bevacizumab-free interval was longer than 3 months in 43% of patients in chemotherapy alone arm and in 50% of patients in the bevacizumab arm. At a median follow-up of 45.3 months, the median progression-free survival was 5.0 months in the chemotherapy group and 6.8 months in the bevacizumab group [adjusted HR = 0.70; 95% confidence interval (CI) 0.52-0.95; stratified log-rank P = 0.010]. Subgroup analyses showed a consistent benefit in all subgroups analyzed and in particular in patients who had continued or reintroduced bevacizumab. An improved overall survival was also observed in the bevacizumab arm (adjusted HR = 0.77; 95% CI 0.56-1.06; stratified log-rank P = 0.043). Responses (RECIST 1.0) were similar in the chemotherapy and bevacizumab groups (17% and 21%; P = 0.573). Toxicity profile was consistent with previously reported data. CONCLUSIONS This study demonstrates that the continuation or the reintroduction of bevacizumab with second-line chemotherapy beyond first progression improves the outcome and supports the use of this strategy in the treatment of mCRC. ClinicalTrials.gov number: NCT00720512.

A pharmacokinetic-based test to prevent severe 5-fluorouracil toxicity

Dihydropyrimidine dehydrogenase (DPD) plays a key role in the catabolism of 5‐fluorouracil (5‐FU) to 5‐fluoro‐5,6‐dihydrouracil (5‐FDHU), and as such, an impairment of DPD has been recognized as an important factor for altered 5‐FU and 5‐FDHU pharmacokinetics, predisposing patients to the development of severe 5‐FU‐associated toxicity. Our objectives were to avoid severe 5‐FU toxicities in patients with greatly impaired 5‐FU and 5‐FDHU pharmacokinetics after the administration of a reduced test dose of 5‐FU and to investigate possible 5‐FU or 5‐FDHU pharmacokinetic parameters of the test dose related to the most common drug toxicities that affect patients after the first cycle of 5‐FU chemotherapy.

MicroRNA Signature in Metastatic Colorectal Cancer Patients Treated With Anti-EGFR Monoclonal Antibodies

BACKGROUND To investigate whether microRNAs are predictive of sensitivity to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies in patients with metastatic colorectal cancer (mCRC). METHODS A total of 183 mCRC cases from 2 independent cohorts (cohort 1: 74 cases; validation cohort: 109 cases) treated with cetuximab or panitumumab were included in the study. MiRNA arrays were analyzed using Agilents miRNA platform. RESULTS The study identified the cluster Let-7c/miR-99a/miR-125b as a signature associated with an outcome different from that of anti-EGFR therapies. In the first cohort, patients with high-intensity signatures had a significantly longer progression-free survival (PFS) (6.1 vs. 2.3 mo; P = .02) and longer overall survival (OS) ( 29.8 vs. 7.0 mo, P = .08) than patients with low-intensity signatures. In the validation cohort, patients with high signature had significantly longer PFS and OS than individuals with low-intensity signatures (PFS 7.8 vs. 4.3 mo, P = .02; OS 12.8 vs. 7.5 mo, P = .02). In the KRAS wild-type population (n = 120), high-intensity signature patients had a significantly longer PFS (7.8 vs. 4.6 mo, P = .016) and longer OS (16.1 vs. 10.9 mo, P = .09) than low-signature individuals, with no difference in KRAS mutated patients. CONCLUSION The MiR-99a/Let-7c/miR-125b signature may improve the selection of patients with KRAS wild-type mCRC as good candidates for anti-EGFR therapy.

Pharmacokinetics of azithromycin in lung tissue, bronchial washing, and plasma in patients given multiple oral doses of 500 and 1000 mg daily.

The present study compares the pharmacokinetics of azithromycin in plasma, lung tissue, and bronchial washing after oral administration of 500 mg (standard dose) versus 1000 mg daily for 3 days. Samples were taken during surgery for lung resection at various time points up to 204 h after the last drug dose, and azithromycin levels were analyzed by HPLC method. Azithromycin was widely distributed within the lower respiratory tract; sustained concentrations of the drug were detectable at the last sampling time (204 h) in lung tissue and bronchial washing, with long terminal half-lives of 132.86 and 74.32 h at 500 mg daily and 133.32 and 70.5 h at 1000 mg daily, respectively. Doubling the drug dose resulted in a remarkable increase in lung area under the curve (AUC, 1318 hx microg g(-1) vs 2502 hx microg g(-1)) and peak tissue concentration (9.13+/-0.53 microg g(-1) vs 17.85+/-2.4 microg g(-1)). In addition to this, enhanced azithromycin penetration from plasma into bronchial secretion and lung tissue was evidenced by the increase in the ratio of AUC(bronchial washing) versus AUC(plasma) (2.96 vs 5.27 at 500 and 1000 mg, respectively) and AUC(lung) versus AUC(plasma) (64.35 vs 97.73 at 500 and 1000 mg, respectively). In conclusion, the exposure of lung and bronchial washing to azithromycin is increased by doubling the dose, which results in favorable pharmacokinetic profile of the drug in the lower respiratory tract.

A Phase I and pharmacokinetic study of irinotecan given as a 7-day continuous infusion in metastatic colorectal cancer patients pretreated with 5-fluorouracil or raltitrexed

Purpose: The purpose is to determine the plasma pharmacokinetics, the maximum-tolerable dose and to preliminary evaluate the antitumor activity of irinotecan administered as a 7-day continuous infusion every 21 days in metastatic colorectal cancer patients pretreated with 5-fluorouracil or raltitrexed. Experimental Design: A total of 13 patients entered the study. Three received irinotecan at 20 mg/m2/day (dose level I), 6 at 25 mg/m2/day (dose level II), and 4 at 22.5 mg/m2/day (dose level III). In 8 patients, plasma levels of irinotecan and its metabolites SN-38 and SN-38 glucuronide (SN-38glu) were measured by high-performance liquid chromatography and main pharmacokinetic parameters, including steady-state concentration, area under the time-concentration curve, and clearance, were calculated and normalized to the dose level of 22.5 mg/m2/day. Results: Dose-limiting toxicity was grade 3–4 diarrhea, which occurred in 4 of 6 patients at dose level II and in 2 of 4 patients at dose level III. Therefore, we defined 22.5 mg/m2/day the maximum-tolerable dose and 20.0 mg/m2/day the recommended dose for Phase II studies. Hematological toxicity was rare. The pharmacokinetic data provided evidence that continuous infusion increased the metabolism of irinotecan to SN-38 with respect to standard 30/90-min administration. Indeed, the steady-state concentration of irinotecan, SN-38, and SN-38glu were 42.7 ± 25.2, 14.9 ± 1.9, and 31.7 ± 3.5 nmol/liter, respectively, and the area under the time-concentration curves of irinotecan, SN-38, and SN-38glu were 6.94 ± 0.41, 1.92 ± 0.30, and 4.23 ± 0.52 hxμmol/liter, respectively. Twelve patients were evaluable for activity, and we observed 3 (25%) partial responses, 2 (17%) minor responses, and 4 (33%) disease stabilizations. Conclusions: The administration of irinotecan as a 7-day continuous infusion every 21 days is feasible with diarrhea being the dose-limiting toxicity; recommended dose for Phase II studies is 20.0 mg/m2/day. The comparison of the present data with those obtained after a standard 30–90 min. i.v. infusion of irinotecan demonstrates that continuous infusion improves the transformation of irinotecan to SN-38 and also results in increased glucuronidation of the active metabolite. Antitumor activity in pretreated metastatic colorectal cancer patients is encouraging.

Variable correlation between 6-mercaptopurine metabolites in erythrocytes and hematologic toxicity: implications for drug monitoring in children with acute lymphoblastic leukemia

Nineteen pediatric patients affected by acute lymphoblastic leukemia (ALL) were examined weekly with respect to 6-mercaptopurine nucleotide (6-MPN) and 6-thioguanine nucleotide (6-TGN) levels in erythrocytes during the course of maintenance treatment with 6-MP 50 mg/m2 per d and results were related to various parameters of bone marrow function to assess, in the same individual, the level of reliability of 6-MP metabolites in predicting a later change in peripheral blood cell counts. Median values for 6-MPN and 6-TGN were 57 and 200 pmol/8 x 10(8) erythrocytes, respectively, as measured by reversed-phase high-performance liquid chromatography (HPLC). 6-TGN levels in erythrocytes were inversely related with white blood cell count (r = -0.463, p < 0.0001, n = 361), absolute neutrophil count (r = -0.386, p < 0.0001, n = 347), erythrocyte (r = -0.354, p < 0.0001, n = 287), and platelet counts (r = -0.24, p < 0.0001, n = 319) in the majority of patients (n = 10-12), while no correlation was found for 6-MPN. In the remaining children, no evidence of correlation was demonstrated between 6-TGN levels and myelotoxicity. The results confirm the role of 6-TGN as the reference cytotoxic metabolite for evaluating the exposure to 6-MP and identifying treatment compliance in ALL children but indicate the limits of a follow-up based solely on metabolite levels and suggest that a more correct approach remains the double monitoring of 6-TGN and blood cell count with differential.

Pharmacokinetics, a main actor in a many‐sided approach to severe 5‐FU toxicity prediction

5-Fluorouracil (5-FU) is a cornerstone in the treatment of many cancers, including colorectal, head and neck, stomach, and breast carcinomas. Recently, 5-FU oral prodrugs, such as capecitabine, have been successfully introduced in cancer therapy, and their clinical use is rapidly and constantly growing. The approach to the prediction of severe toxicities due to 5-FU (and its oral prodrugs) has been matter of debate for many years and still remains a hot topic in oncology. The efforts in this field are almost entirely focused on the analysis of dihydropyrimidine dehydrogenase (DPD) gene mutations and of peripheral blood mononuclear cell (PBMC) DPD activity. For these reasons, in this letter we would like to suggest a novel, rational diagnostic algorithm that, integrating the analysis already available, is centred on the use of 5-FU and 5-fluoro-5,6-dihydrouracil (5-FDHU) pharmacokinetics as a tool to prevent severe and life-threatening 5-FU toxicities in patients with impaired 5-FU metabolism. DPD plays a pivotal role in the metabolism of 5-FU [1] and, as such, a deficiency of DPD has been recognized as an important risk factor, predisposing patients to the development of severe toxicity. Numerous genetic [2, 3] and phenotypic (i.e. DPD activity, breath test or plasma dihydrouracil/uracil ratio) [4–6] approaches have been proposed to prevent life-threatening toxicities, and, in our opinion, the time has come to develop an integrated approach to this clinically relevant issue. The screening of DPYD mutations [3] and of peripheral blood mononuclear cell DPD activity [7] in patient candidates for 5-FU treatment has been proposed as a routine approach to identify severely reduced DPD activity. However, both the genotype and phenotype options have some advantages but also limitations, as comprehensively pointed out by Ezzeldin and Diasio [8]. In order to identify all patients with impaired 5-FU clearance, no matter which type of DPYD mutations or DPD activity in PBMC compartment they have, in the last decade some authors have suggested a phenotypic approach of screening, looking at the systemic 5-FU catabolism and measuring the pharmacokinetic parameters of the parental drug and of its principal metabolite 5-FDHU [9–11]. As an example, our group has recently proposed the administration of a 5-FU test dose in patient candidates for 5-FU chemotherapy to calculate the pharmacokinetic parameters, such as 5-FU clearance and t1/2β or 5-FDHU Cmax, Tmax and t1/2β, that could be profoundly altered in the presence of an impaired systemic clearance [12]. Indeed, our study revealed markedly impaired 5-FU and 5-FDHU kinetics in some patients, as previously shown for patients with severe toxicities in other published studies [13], suggesting a possible profound alteration of 5-FU metabolism; owing to the detection of this abnormality, these subjects were not given a potentially life-threatening standard dose of 5-FU. Moreover, based on our experimental data, statistically significant relationships have been demonstrated between 5-FDHU pharmacokinetic parameters and moderate to severe degrees of three major dose-limiting toxicities related to 5-FU treatments (stomatitis, diarrhoea and neutropenia) after the first cycle of 5-FU standard therapy. In particular, a prolonged 5-FDHU t1/2β and a 5-FDHU Tmax higher than the median value of our population (30 min) could help identify patients at risk of developing moderate to severe neutropenia or diarrhoea [12]. The 5-FU test dose can be easily performed in a hospital setting with a clinical pharmacology unit such as those usually present in medium- or large-sized university hospitals of Western countries. In particular, an organized regional network among major clinical oncology units may allow screening patients within 2–3 days. The analysis can be available in a few days and the patients can be ready to start the 5-FU therapy at standard or reduced dose in the following week when 5-FU and 5-FDHU kinetic parameters are available. Based on this clinical experience, we detail the principles that should guide the decision-making process regarding the prevention of 5-FU severe toxicity and propose a diagnostic algorithm (Figure 1) in order to screen candidate patients to fluoropyrimidine therapy. In the suggested diagnostic algorithm (Figure 1), the predictive 5-FU test dose could be regarded as a triage test, allowing detection of the fraction of patients with normal, impaired or absent fluoropyrimidine metabolism. Other analyses, such as DPD genotyping or even DPD PBMC activity, could be used later as add-on tests and, limited to the still undiagnosed subgroup, to detect those degrees of enzyme activity impairment suitable for possible reduction of 5-FU dose or different treatments. Overall, the published data strongly suggest the use of a diagnostic algorithm based on the sequential application of a 5-FU pharmacokinetic test followed by DPD genotyping and activity in order to make a highly probable diagnosis of altered 5-FU metabolism. Moreover, the application of this model could result in a consistent reduction of costs and morbidity, by limiting genotyping and PBMC DPD activity analysis to only selected subgroups of patients. Figure 1 Suggested diagnostic flow chart for the detection of patients with impaired fluoropyrimidine metabolism. 5-FU, 5-fluorouracil; 5-FDHU, 5-fluoro-5,6-dihydrouracil; DPD, dihydropyrimidine dehydrogenase; PBMC, peripheral blood mononuclear cell; PK, pharmacokinetics; ... In conclusion, the published clinical experience suggests that an integrated approach based on pharmacokinetic analysis combined with DPD genotyping and/or phenotyping seems to be a safer strategy for optimizing the administration of 5-FU and its oral prodrugs, which remain major drugs used extensively in clinical oncology.

Idarubicin and idarubicinol effects on breast cancer multicellular spheroids.

Abstract Despite extensive preclinical evaluation in several experimental models, no studies have determined the effect of idarubicin and its metabolite idarubicinol on multicellular spheroids, a model which mimics the microregions of solid tumors. The principal aim of the present study was to investigate the in vitro cytotoxicity of idarubicin and its metabolite idarubicinol on MCF-7 breast cancer cells growing as monolayers or multicellular spheroids and to evaluate the influence of the length of exposure on the cytotoxic effect of both drugs. Cytoxicity was evaluated on monolayer and spheroid cultures exposed to idarubicin and idarubicinol 0.01-1000 ng/ml for 24 h or treated for 6, 12, 24 and 48 h to 100 ng/ml of both drugs. The IC50 of idarubicin and idarubicinol were 3.3±0.4 and 3.6±0.7 ng/ml, respectively, on MCF-7 monolayers and 7.9±1.1 and 5.3±0.7 ng/ml in multicellular spheroids, respectively. The antiproliferative effects of 100 ng/ml idarubicin and idarubicinol on MCF-7 spheroids was characterized by a marked time-dependence, which was less evident on MCF-7 growing as monolayer. In conclusion, the present experimental data demonstrate, for the first time, that idarubicin and idarubicinol have significant cytotoxic activity against multicellular spheroids, comparable to the antiproliferative effects on monolayer cells. In contrast, spheroids displayed substantial resistance after short exposure times that was not present in the two dimensional cultures.