Jérôme Alexandre

Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?

Increased generation of reactive oxygen species (ROS) and an altered redox status have long been observed in cancer cells, and recent studies suggest that this biochemical property of cancer cells can be exploited for therapeutic benefits. Cancer cells in advanced stage tumours frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it might be possible to preferentially eliminate these cells by pharmacological ROS insults. However, the upregulation of antioxidant capacity in adaptation to intrinsic oxidative stress in cancer cells can confer drug resistance. Abrogation of such drug-resistant mechanisms by redox modulation could have significant therapeutic implications. We argue that modulating the unique redox regulatory mechanisms of cancer cells might be an effective strategy to eliminate these cells.

Safety and Pharmacokinetics of Escalated Doses of Weekly Intravenous Infusion of CCI-779, a Novel mTOR Inhibitor, in Patients With Cancer

PURPOSE To establish the safety, tolerability, and pharmacokinetic parameters of CCI-779, a selective inhibitor of the mammalian target of rapamycin, in patients with advanced cancer. PATIENTS AND METHODS Using a modified continuous reassessment method, we performed a phase I with pharmacokinetic study of CCI-779 given as a weekly 30 minutes intravenous (I.V.) infusion. RESULTS Twenty-four patients received CCI-779 at doses ranging 7.5 to 220 mg/m(2). No immunosuppressive effect was reported. Dose-limiting thrombocytopenia occurred in two patients at 34 or 45 mg/m(2). At 220 mg/m(2), dose-limiting toxicities consisted of manic-depressive syndrome, stomatitis, and asthenia in two of nine patients, preventing further dose escalation. The most frequent drug-related toxicities were acne-like, maculopapular rashes and mucositis or stomatitis. All toxicities were reversible on treatment discontinuation. Maximum concentration and area under the concentration-time curve increase sub-proportionally with dose. Mean steady-state volume of distribution ranged from 127 to 385L. Sirolimus was a major metabolite (metabolite-to-parent ratio range, 2.5 to 3.5). Whole blood clearance was nonlinear, ranging from 19 to 51 L/h (34 to 220 mg/m(2)). Variability predicted with flat doses appears comparable with data based on body-surface area-normalized treatment. Partial responses were observed in one patient with renal clear-cell carcinoma and in one patient with breast adenocarcinoma. CONCLUSION CCI-779 displayed no immunosuppressive effects with manageable and reversible adverse events at doses up to 220 mg/m(2), the highest dose tested. Based on our results, weekly doses of 25, 75, and 250 mg CCI-779 not based on classical definitions of maximum-tolerated dose are being tested in phase II trials in patients with breast and renal cancer.

Phase II Study of Ecteinascidin-743 in Advanced Pretreated Soft Tissue Sarcoma Patients

PURPOSE A multicenter phase II study evaluating efficacy, safety, and pharmacokinetics of ecteinascidin-743 (ET-743) in pretreated advanced soft tissue sarcoma patients. PATIENTS AND METHODS Patients received ET-743 1,500 microg/m(2) (24-hour intravenous infusion) every 3 weeks (group 1, 26 patients with one to two prior single agents or one previous combination chemotherapy; group 2, 28 patients with three or more prior single agents or two or more previous combination chemotherapies). Results Patients (30 women, 24 men) had a median age of 48 years (range, 22 to 71 years); 41% had leiomyosarcoma (eight of 22 of uterine origin), a median of two involved organs (range, one to four), and 93% had documented progressive disease at study entry. Patients received a median of three cycles (range, one to 20); 28% received six or more cycles. Fifty-two patients were assessable for response (WHO criteria): two partial responses, four minor responses, and nine with stable disease (> or = 6 months). Three patients were rendered tumor free after surgery. Median progression-free survival was 1.9 months (range, 0.69 to 17.90 months); 24% of patients were progression free at 6 months. Median survival was 12.8 months, with 30% of patients alive at 2 years. Four patients withdrew because of treatment-related toxicity. Two treatment-related deaths occurred (renal failure and febrile neutropenia, and rhabdomyolysis and decompensated cirrhosis, respectively) that were probably related to protocol eligibility violations. Reversible grade 3 to 4 AST or ALT occurred in 50% of patients and grade 3 to 4 neutropenia occurred in 61% of patients, with six episodes of febrile neutropenia. Nausea, vomiting, and asthenia were prevalent but mild and manageable. CONCLUSION With a 4% overall response rate (95% CI, 0.5 to 12.8) and an 11% rate of third-party-verified tumor regression (overall response rate + minor response), ET-743 has a 24% 6-month disease progression control rate, confirming evidence of antitumoral activity and a manageable safety profile in patients experiencing disease progression with pretreated soft tissue sarcoma.

Accumulation of hydrogen peroxide is an early and crucial step for paclitaxel-induced cancer cell death both in vitro and in vivo.

Intracellular events following paclitaxel binding to microtubules that lead to cell death remain poorly understood. Because reactive oxygen species (ROS) are involved in the cytotoxicity of anticancer agents acting through independent molecular targets, we explored the role of ROS in paclitaxel cytotoxicity. Within 15 min after in vitro exposure of A549 human lung cancer cells to paclitaxel, a concentration‐dependent intracellular increase in O°2− and H2O2 levels was detected by spectrofluorometry. Addition of N‐acetylcysteine (NAC) or glutathione, two H2O2 scavenger, induced a 4‐fold increase in paclitaxel IC50. Delaying NAC co‐incubation by 4 hr, resulted in a 3‐fold reduction in cell protection. The glutathione synthesis inhibitor, buthionine sulfoximine significantly increased paclitaxel cytotoxicity and H2O2 accumulation, but did not modify O°2− levels. Co‐incubation with diphenylene iodonium suggested that paclitaxel induced‐O°2− production was in part associated with increased activity of cytoplasmic NADPH oxidase. Concomitant treatment with inhibitors of caspases 3 and 8 increased cell survival but did not prevent the early accumulation of H2O2. To evaluate the role of ROS in paclitaxel antitumoral activity, mice were injected with LLC1 lung cancer cells and treated with paclitaxel i.p. and/or NAC. The antitumoral activity of paclitaxel in mice was abolished by NAC. In conclusion, the accumulation of H2O2 is an early and crucial step for paclitaxel‐induced cancer cell death before the commitment of the cells into apoptosis. These results suggest that ROS participate in vitro and in vivo to paclitaxel cytotoxicity.

Novel Action of Paclitaxel against Cancer Cells: Bystander Effect Mediated by Reactive Oxygen Species

Generation of reactive oxygen species (ROS) has been observed in cancer cells treated with paclitaxel, but the underlying mechanisms and therapeutic implications remain unclear. In the present study, we showed that paclitaxel promoted ROS generation through enhancing the activity of NADPH oxidase (NOX) associated with plasma membranes. Treatment of breast cancer cells caused an increased translocation of Rac1, a positive regulatory protein of NOX, to the membrane fraction. The paclitaxel-induced ROS generation occurred rapidly within several hours of drug exposure, with O(2)(-) and H(2)O(2) accumulation mainly outside the cells while the intracellular ROS remained unchanged. Importantly, the increase in extracellular ROS caused lethal damage to the bystander cancer cells not exposed to paclitaxel, as shown by two different methods using coculture systems where the bystander cells were differentiated from the paclitaxel-treated cells by fluorescent or radioactive labeling. This cytotoxic bystander effect was also observed with other microtubule-targeted agents vincristine and taxotere but not with 5-fluorouracil or doxorubicin. This toxic bystander effect was enhanced by CuZnSOD that converts O(2)(-) to H(2)O(2) and was abolished by a catalase that eliminates H(2)O(2). Furthermore, paclitaxel was able to induce an almost complete inhibition of proliferation of the bystander cells in the coculture system. Our study revealed a novel mechanism by which paclitaxel induces toxic bystander effect through generation of extracellular H(2)O(2) from the membrane-associated NOX. This may contribute to the potent anticancer activity of paclitaxel and provide a novel basis to improve the clinical use of this important drug.

Sorafenib-Induced Hepatocellular Carcinoma Cell Death Depends on Reactive Oxygen Species Production In Vitro and In Vivo

Sorafenib is presently the only effective therapy in advanced hepatocellular carcinoma (HCC). Because most anticancer drugs act, at least in part, through the generation of reactive oxygen species, we investigated whether sorafenib can induce an oxidative stress. The effects of sorafenib on intracellular ROS production and cell death were assessed in vitro in human (HepG2) and murine (Hepa 1.6) HCC cell lines and human endothelial cells (HUVEC) as controls. In addition, 26 sera from HCC patients treated by sorafenib were analyzed for serum levels of advanced oxidation protein products (AOPP). Sorafenib significantly and dose-dependently enhanced in vitro ROS production by HCC cells. The SOD mimic MnTBAP decreased sorafenib-induced lysis of HepG2 cells by 20% and of Hepa 1.6 cells by 75% compared with HCC cells treated with 5 mg/L sorafenib alone. MnTBAP significantly enhanced by 25% tumor growth in mice treated by sorafenib. On the other hand, serum levels of AOPP were higher in HCC patients treated by sorafenib than in sera collected before treatment (P < 0.001). An increase in serum AOPP concentration ≥0.2 μmol/L chloramine T equivalent after 15 days of treatment is a predictive factor for sorafenib response with higher progression free survival (P < 0.05) and overall survival rates (P < 0.05). As a conclusion, sorafenib dose-dependently induces the generation of ROS in tumor cells in vitro and in vivo. The sera of Sorafenib-treated HCC patients contain increased AOPP levels that are correlated with the clinical effectiveness of sorafenib and can be used as a marker of effectiveness of the drug. Mol Cancer Ther; 11(10); 2284–93. ©2012 AACR.

Relationship between GSTP1 Ile105Val polymorphism and docetaxel-induced peripheral neuropathy: clinical evidence of a role of oxidative stress in taxane toxicity

BACKGROUND Glutathione-S-transferases (GST) regulate the cellular response to oxidative stress. We previously highlighted the importance of oxidative stress in taxane toxicity and therefore investigated the relationship between the GST isoforms M1, T1 and P1 gene polymorphisms and docetaxel (Taxotere)-induced peripheral neuropathy (DIPN). PATIENTS AND METHODS The GSTM1 (null), GSTT1 (null) and GSTP1 (Ile(105)Val and Ala(114)Val) polymorphisms were determined in a cohort of cancer patients treated with docetaxel and entered in a clinical trial database. The relationship between GST polymorphisms and grade > or = 2 DIPN as primary end point was studied. RESULTS Fifty-eight patients (median age 61 years) received a total of 261 cycles of docetaxel given as single agent. Patients with GSTP1 (105)Ile/(105)Ile genotype had a higher risk of developing a grade > or = 2 DIPN than did those with other GSTP1 genotypes (8 of 27 versus 2 of 31, respectively, odds ratio 6.11; 95% confidence interval 1.17-31.94; P = 0.03). In multivariate analysis, grade > or = 2 DIPN was strongly correlated with GSTP1 (105)Ile/(105)Ile genotype (P = 0.01) and the number of cycles (P = 0.03). CONCLUSION We found a significant correlation between GSTP1 (105)Ile/(105)Ile genotype and the development of grade > or = 2 DIPN. This finding strongly suggests a role of oxidative stress in the pathophysiology of DIPN.

High-dose intrathecal trastuzumab for leptomeningeal metastases secondary to HER-2 overexpressing breast cancer

We are facing an increasing incidence of brain and leptomeningeal metastases (LM) during the history of patients with HER-2-overexpressing breast cancer. This clinical issue is due to the neurotropism of HER-2-overexpressing breast cancer cells, combined with the high antitumoral activity of systemic administrations of trastuzumab but without preventive effect in the brain and leptomeninges. Indeed, trastuzumab is a monoclonal IgG1 antibody to HER-2, which poorly reaches the cerebrospinal fluid (CSF) when given i.v. [1]. Intrathecal (IT) administration of trastuzumab has been scarcely tested [2–5] in patients with LM occurring after a systemic complete remission induced by i.v. trastuzumab (Table 1). Therefore, we eagerly need to accumulate experience on the use of IT trastuzumab in this setting. We report here the case of a patient with HER-2-overexpressing breast cancer who developed LM. We obtained a long-lasting control of the disease progression using high doses of IT trastuzumab. In April 2001, a 55-year-old patient was diagnosed with a breast cancer (T1N0M0, ER+). She was initially treated by surgery, radiotherapy and tamoxifen. Eighteen months later, she developed multiple liver metastases. The breast cancer was tested for HER-2 and was found positive (3+ by immunohistochemistry). She received docetaxel plus epirubicin, then paclitaxel plus trastuzumab and achieved a complete remission. One year later, headaches and ataxia revealed massive involvement of the brain by multiple metastases. A whole-brain irradiation allowed achieving a complete remission of the brain metastases. However, while under i.v. trastuzumab, 15 months after the liver remission and 9 months after the brain remission, she developed midback pain, cerebellar ataxia and headaches, leading to a clinical diagnosis of LM. Cerebral and spinal MRI scans revealed pericerebellar and cervical leptomeningeal foci, consistent with carcinomatous involvement. Initial CSF examination was normal, except for proteins of 0.68 g/l. Computed tomography scans confirmed that she had no recurrence of liver and brain metastases. The patient underwent daily oral corticosteroids and was informed of the poor prognosis for this condition. She refused a conventional treatment by IT high-dose methotrexate as described by Fizazi et al. [6]. Hence, after she gave her informed consent, she was administered weekly IT trastuzumab 20 mg (20 mg being the highest IT dose experienced in humans [2–5]) by lumbar puncture. The first administration was well tolerated and thereafter we carried out a dose escalation every week: 40 mg, then 100 mg weekly, for a total of six cycles of IT trastuzumab over 6 weeks (20 mg · 1, 40 mg · 1, 100 mg · 4). The patient experienced striking clinical neurological improvement, with complete disappearance of headaches and ataxia after 2 weeks of treatment. The changes in CSF are summarized in Table 2. It is noteworthy that the CSF proteins increased with repeated administrations. The cerebral magnetic resonance imaging (MRI) at 6 weeks showed a stable disease. However, 2 months later, the patient’s status worsened due to progression of brain metastases in previously irradiated areas, without evidence of meningeal disease progression. Despite high doses of i.v. corticosteroids, she finally developed intracranial hypertension and died of brain metastases 5 months later, 7 months from the diagnosis of LM. The use of IT IgG1 mAbs is supported by the knowledge that they poorly cross the blood–brain barrier when given i.v. [1, 7], making the CNS a sanctuary for cancer cells. To our knowledge, this case is the first report of a safe administration of IT trastuzumab at high doses (100 mg weekly). The major clinical improvement observed in our patient was not reflected by changes in CSF proteins, whereas MRI scans showed a stable disease. We assume that the discrepancy between clinical improvement and CSF proteins could be due to the accumulation of high doses of trastuzumab in the CSF. However, monitoring of trastuzumab CSF levels as described letters to the editor Annals of Oncology

An Observational Study of Bevacizumab-Induced Hypertension as a Clinical Biomarker of Antitumor Activity

BACKGROUND Hypertension is a common toxicity of bevacizumab, but the frequency of assessment of blood pressure and standardized grading remain to be defined. This study aimed to describe the incidence of bevacizumab-induced hypertension and factors associated with its development, then to retrospectively assess its relation with activity. PATIENTS AND METHODS One hundred nineteen patients with advanced or metastatic non-small cell lung cancer, colorectal cancer, or ovarian cancer receiving bevacizumab (2.5 mg/kg per week) and chemotherapy were eligible for this analysis. Blood pressure was measured at home twice daily according to international guidelines, and graded according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC), version 3.0, and the European Society of Hypertension (ESH) criteria. RESULTS Home-based measurements detected significantly more cases of hypertension than in-clinic measurements did, according to the ESH criteria (54.6% versus 24.4%; p < .001) or the NCI-CTC (42.9% versus 22.7%; p = .0015). Very early hypertension (within 42 days, according to the ESH criteria) but not hypertension (occurring at any time during treatment period) was predictive of response (p = .0011 and p = .26, respectively). CONCLUSIONS Our preliminary results indicate that home-based measurement and grading according to the ESH criteria represents a reliable method to detect bevacizumab-induced hypertension. Whether hypertension is a biomarker of bevacizumab activity remains to be determined in a prospective study.

Weekly paclitaxel as a single agent or in combination with carboplatin or weekly topotecan in patients with resistant ovarian cancer: the CARTAXHY randomized phase II trial from Groupe d’Investigateurs Nationaux pour l’Etude des Cancers Ovariens (GINECO)

BACKGROUND Platinum rechallenge or weekly topotecan in combination have not been evaluated in randomized trials for resistant recurrent ovarian cancer (ROC). METHODS Patients with ROC after first- or second-line treatment including a platinum and taxane and progression within 6 months were randomized to weekly paclitaxel (wP, 80 mg/m(2)/week) alone or in combination with carboplatin (C, area under the curve of 5 mg/ml/min every 4 weeks) or weekly topotecan (wT, 3 mg/m(2)/week). Primary end point was progression-free survival (PFS) comparing wP and combination therapy. RESULTS Patients (n = 165) received a median three cycles in each arm. Nonhematologic toxicity was not different, except increased hypersensitivity reactions with wP + C. Grade 3-4 hematologic toxic effects with wP, wP + C, and wP + wT, respectively, were neutropenia in 13%, 54%, and 42%; febrile neutropenia in 0%, 4%, and 5%; and anemia in 6%, 19%, and 29%. Response rates were 35%, 37%, and 39%, and median PFS times were 3.7, 4.8, and 5.4 months, respectively. PFS was not significantly different among the treatment arms [hazard ratio (HR) 0.922; 95% confidence interval (CI) 0.765-1.111; P = 0.46] or between monotherapy and combination therapy (HR 0.951; 95% CI 0.686-1.318; P = 0.76). CONCLUSIONS Combination chemotherapy in platinum-resistant ROC was more toxic than weekly paclitaxel and did not significantly prolong PFS.