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PDF file - 579K, S1. Validation of the MDA-MB-435s cell system expressing luciferase gene and expressing or not KCNN3 gene. S2. SK3 knockdown reduced the development of metastases in cancer cell xenograft models. A) SK3 knockdown reduced the metastatic score in cancer cell xenograft model. S3.Role of Orai1 in the constitutive SK3-dependent calcium entry and SK3+ cell migration. S4. Effect of Ohmline on Orai1 and SK3 expression and on constitutive calcium entry. S5. SK3 protein was expressed in smooth muscle cells of urinary bladder detrusor and myometrium and in primary tumour form mice grafted with SK3+ cells 16 weeks post grafted.

International audience; The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca2+ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca2+ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca2+ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca2+ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.

In a previous pilot study, we showed that polyunsaturated n-3 fatty acids of breast adipose tissues were associated with breast cancer multifocality. In the present study, we investigated biochemical, clinical and histological factors associated with breast cancer focality in a large cohort of women with positive hormone-receptors tumors. One hundred sixty-one consecutive women presenting with positive hormone-receptors breast cancer underwent breast-imaging procedures including a Magnetic Resonance Imaging prior to treatment. Breast adipose tissue specimens were collected during surgery of tumors. A biochemical profile of breast adipose tissue fatty acids was established by gas chromatography. Clinicopathologic characteristics were correlated with multifocality. We assessed whether these factors were predictive of breast cancer focality. We found that tumor size (OR = 1.06 95%CI [1.02-1.09], p 0.001) and decreased levels in breast adipose tissue of long-chain polyunsaturated n-3 fatty acids (OR = 0.11 95%CI [0.01-0.98], p = 0.03), were independent predictive factors of multifocality. Low levels of long chain polyunsaturated n-3 fatty acids in breast adipose tissue appear to contribute to breast cancer multifocality. The present results reinforce the link between dietary habits and breast cancer clinical presentation.

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Objective Since it is thought that breast adipose tissue could influence breast cancer clinical presentation, we wanted to characterize specifically the relationship between breast adipose tissue fatty acid profile and Inflammatory Breast cancer (IBC). Methods Two hundred thirty-four women presenting with breast cancer were managed in our centre between January 2009 and December 2011. Breast adipose tissue specimens were collected during breast surgery. We established the biochemical profile of adipose tissue fatty acids (FA) by gas chromatography and assessed whether there were differences in function of the presence of breast inflammation or not. Results We found that IBC was associated with decreased levels in breast adipose tissue of eicosapentaenoic acid (EPA), one of the two main polyunsaturated n-3 fatty acids (n-3 PUFA) of marine origin, but also with decreased levels of Gamma Linolenic acid (GLA). Inversely, an increase in palmitic acid levels was associated with IBC. Conclusion These differences in lipid content may contribute to the occurrence of breast cancer inflammation.

International audience; The fatty acids composition of adipose tissue may provide information on the nutritional part of the risk or evolution of breast cancer. To determine whether 1H NMR of adipose tissue provides information on the nature of the diet consumed, a dietary intervention with increasing percentage of polyunsaturated n-3 docosahexaenoic acid (DHA 22:6n-3, provided as DHASCO oil) was applied to a rat model of N-nitroso-N-methylurea-induced mammary tumors. Spectra of the lipid extracts were obtained from adipose tissues in five groups of Sprague–Dawley rats fed with a diet containing 7% peanut/rapeseed enriched with 8% (w/w) of an oil without (palm oil) or with low (1%), moderate (3%), or high (8%) DHASCO content. A control group received a basal diet with 15% peanut/rapeseed representative of the “Western” diet. After 5 months of those five controlled diets, adipose tissue was collected for analysis of the lipid extract using both 1H NMR analysis on an 11.7 T spectrometer and gas chromatography considered as gold standard. 1H NMR analysis showed a dose-dependent increase in DHA in the lipid extract of adipose tissues and a commensurate decrease in n-6 polyunsaturated fatty acids in the three DHA groups, which allowed one to follow n-6/n-3 ratio changes. The highest n-6/n-3 ratio was observed in the control Western diet group compared to the other diet groups. The integrated spectral regions showed separation between groups, thereby documenting a specific NMR lipid profile corresponding to each dietary intervention. Those diet-dependent NMR lipid profiles were consistent with that obtained with gas chromatography analyses of the same samples. This study is a proof of concept highlighting the potential use of the 1H NMR approach to evaluate dietary intervention in biopsies of adipose tissues.

Purpose: Taxane-induced peripheral neuropathy is a common side effect induced by anticancer agents, and no drug capable of preventing its occurrence or ameliorating its long-term course has been identified. The physiology of taxane neuropathy is not clear, and diverse mechanisms have been suggested, with ion channels regulating Ca2+ homeostasis appearing good candidates. The calcium-activated potassium channel SK3 is encoded by the KCNN3 gene, which is characterized by a length polymorphism due to variable number of CAG repeats. Experimental Design: To study the influence of the polymorphism of CAG motif repeat of KCNN3 on the development of taxane-induced neuropathy, we evaluated 176 patients treated with taxanes for breast cancer. In parallel, we measured Ca2+ entry using Fura2-AM dye in HEK cells expressing short versus long CAG alleles of KCNN3. Results: In the current study, we report that in the presence of docetaxel, Ca2+ entry was significantly increased in cells expressing short versus long CAG alleles of SK3 and that a SK3-lipid blocker inhibits this effect. We found that patients carrying a short KCNN3 allele exhibited significantly increased incidence of taxane neuropathy compared with those carrying longer alleles. Conclusions: The clinical implication of these findings is that KCNN3 polymorphism may increase patient susceptibility to taxane neurotoxicity and that the use of SK3 blockers during taxanes’ administration may represent an interesting approach for the prevention of this neurotoxicity. Clin Cancer Res; 24(21); 5313–20. ©2018 AACR.

Background: Long chain n-3 Polyunsaturated Fatty Acids (n-3 PUFA) of marine origin (docosahexaenoic acid, DHA and eicosapentaenoic acid, EPA) have been shown to increase sensitivity of cell lines to anthracyclins or taxanes. Preclinical studies and a phase II clinical trial have shown that increasing DHA level in the body tissues through a dietary supplementation improves chemotherapy efficacy. We have set up a phase III controlled, double blind study to determine whether a dietary supplementation with n-3 PUFA would increase PFS in patients receiving chemotherapy for metastatic breast cancer (MBC). Methods: Inclusion criteria were patients with a ductal or lobular breast carcinoma, positive hormone receptors, Her2 (-), who have developed visceral metastasis, and were due to receive a 1st or 2d line chemotherapy. Dietary intervention was carried out during chemotherapy. Patients had to take daily at each meal a can* (medical food) containing either fish oil (1.56 g/d of DHA and 2.64 g/d EPA, experimental arm) or coprah oil (short chains fatty acids, control arm). Principal endpoint was PFS, secondary were objective response, overall survival, dietary tolerance to cans, lipids plasma levels of n-3 PUFA. Results: Sixty five patients with MBC have been prospectively enrolled in the DHALYA trial (NTC01548534). The first 45 patients who completed chemotherapy along with dietary supplementation are evaluable. Plasma fatty acid level was measured at baseline, at C1 after 10 days loading dose, at C3, and at C6 or at withdrawal. Level of EPA and DHA increased in half of the patients, thus allowing a putative allocation into either arm: arm A with induced elevation of PUFA levels (N=22), and arm B without any change in PUFA levels (N=23). No difference was observed in the quality of the dietary intervention (number of cans, duration) among arms. Distribution of patients according to demographics (age, menopausal status, histology, SBR grade), or type or quality of chemotherapy received (anthracycline- or taxane-based, number of cycles, length) was similar between arms. However a significantly greater proportion of patients with poor prognosis at staging (larger tumor size and greater axillary lymph nodes involvement) were observed in arm A. There was no difference in side effects (grade ≥2) between arms. In terms of efficacy, median PFS was 14.3 (arm A) and 12.5 (arm B) months, not statistically different yet. Conclusion: A dietary intervention targeted on marine-derived PUFA during systemic chemotherapy for MBC is safe and feasible. A longer follow-up is required in order to know whether chemotherapy efficacy can be increased. This study was supported by a grant from the French Ministry of Health, PHRC-11-153. *Cans were graciously provided by Nutrialys Medical Nutrition, Saint Grégoire, France. Citation Format: Philippe Bougnoux, Jacques Bonneterre, Anne Mercier-Blas, Patrick Soulié, Hélène Simon, Franck Priou, Christine Piprot-Choffat, Christelle Levy, Caroline Goupille, Virginie Berger. Modification of the response to chemotherapy of HER2 negative metastatic breast cancer by lipids of marine origin: A controlled, randomized, double blind dietary supplementation trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-13-08.

Cancer-induced cachexia describes the progressive skeletal muscle wasting associated with many cancers leading to shortened survival time in cancer patients. We previously reported that cardiolipin content and energy-wasting processes were both increased in liver mitochondria in a rat model of peritoneal carcinosis (PC)-induced cachexia. To increase the understanding of the cellular biology of cancer cachexia, we investigated the involvement of adenine nucleotide translocator (ANT) in mitochondrial energy-wasting processes in liver mitochondria of PC and pair-fed control rats and its interactions with cardiolipin in isolated liver mitochondria from healthy rats exposed to cardiolipin-enriched liposomes. We showed in this study that functional ANT content was decreased in liver mitochondria from PC rats but without any effects on the efficiency of ATP synthesis. Moreover, non-phosphorylating energy wasting was not affected by saturating concentrations of carboxyatractylate (CAT), a potent inhibitor of ANT, in liver mitochondria from PC rats. Decreased efficiency of ATP synthesis was found in normal liver mitochondria exposed to cardiolipin-enriched liposomes, with increased non-phosphorylating energy wasting, thus mimicking mitochondria from PC rats. However, the functional ANT content in these cardiolipin-enriched mitochondria was unchanged, although non-phosphorylating energy wasting was reduced by CAT-induced inhibition of ANT. Finally, non-phosphorylating energy wasting was increased in cardiolipin-enriched mitochondria with substrates for complexes 1 and 2, but not for complex 4. In conclusion, increased energy wasting measured in liver mitochondria from rats with cancer cachexia is dependent on cardiolipin but independent of ANT. Interactions between ANT and cardiolipin are modified when cancer cachexia occurs.

SK3 channel mediates the migration of various cancer cells. When expressed in breast cancer cells, SK3 channel forms a complex with Orai1, a voltage-independent Ca(2+) channel. This SK3-Orai1 complex associates within lipid rafts where it controls a constitutive Ca(2+) entry leading to cancer cell migration and bone metastases development. Since cAMP was found to modulate breast cancer cell migration, we hypothesized that this could be explained by a modulation of SK3 channel activity. Herein, we study the regulation of SK3 channel by the cAMP-PKA pathway and the consequences for SK3-dependent Ca(2+) entry and cancer cell migration. We established that the beta-adrenergic receptor agonist, isoprenaline, or the direct adenylyl cyclase activator forskolin alone or in combination with the PDE4 inhibitor, CI-1044, decreased SK3 channel activity without modifying the expression of SK3 protein at the plasma membrane. Forskolin and CI-1044 reduced the SK3-dependent constitutive Ca(2+) entry and the SK3-dependent migration of MDA-MB-435s cells. PKA inhibition with KT 5720 reduced: (1) the effect of forskolin and CI-1044 by 50 % on Ca(2+) entry and (2) SK3 activity by inhibiting the serine phosphorylation of SK3. These cAMP-elevating agents displaced Orai1 protein outside lipid rafts in contrast to SK3, which remained in the lipid rafts fractions. All together, these results show that activation of the cAMP-PKA pathway decreases SK3 channel and SK3-Orai1 complex activities, leading to a decrease in both Ca(2+) entry and cancer cell migration. This work supports the potential use of cAMP-elevating agents to reduce cancer cell migration and may provide novel opportunities to address/prevent bone metastasis.

Purpose/Objectives Few data are available regarding the safety of the concurrent combination of bevacizumab with adjuvant radiotherapy (RT) in breast cancer, especially in terms of late toxicity. The aim of this study was to determine early and late loco-regional toxicities among patients with non-metastatic breast cancer treated with this combination. Materials/Methods In our prospective and descriptive study, we analyzed loco-regional toxicities of adjuvant RT in patients with non-metastatic breast cancer receiving either concurrent bevacizumab or not in the randomized trial BEATRICE. Early and late toxicities were assessed by the Common Terminology Criteria for Adverse Events (v3.0). Evaluation was done during RT and 12 months after the end of RT. All patients provided written informed consent before enrollment. Statistical analysis was performed to analyze toxicity between the two groups. Results From September 2007 to July 2009, we included 84 patients from the randomized trial BEATRICE which evaluate the efficacy and safety of the addition of bevacizumab to standard adjuvant therapy in patients with triple negative breast cancer; 39 women received an adjuvant RT with concurrent bevacizumab and 45 women received an adjuvant RT alone. Evaluation at 12 months was available for all the patients. All patients had a triple negative non-metastatic breast cancer and had an adjuvant chemotherapy then RT. Among patients receiving concurrent bevacizumab with RT, a total of 35 patients (90%) achieved a whole breast irradiation (median dose: 50 Gy) with a boost in the surgical bed (median dose: 16 Gy) and 4 patients (10%) had a post mastectomy RT (median dose 50 Gy); lymph node RT was performed in 19 patients (49%) with internal mammary chain RT in 12 patients (31%). Mean time of bevacizumab treatment was 11.7 months [2.1-12.6] and mean total dose of bevacizumab was 15000 mg [3330-28080]. Among patients receiving RT alone, 38 patients (84%) achieved a whole breast irradiation (median dose: 50 Gy) with a boost in the surgical bed (median dose: 16 Gy) and 7 patients (16%) had a post mastectomy RT (median dose 50 Gy); lymph node RT was performed in 21 patients (47%) with internal mammary chain RT in 14 patients (31%). Radiation treatment parameters were not significantly different between the two groups. Incidence of acute grade 3 dermatitis was 10% in patients receiving bevacizumab associated with RT and 6% in patients receiving RT alone without significant difference. One year after the end of RT, the most common late toxicities in the group receiving bevacizumab and RT were grade 1-2 pain (18%), grade 1-2 fibrosis (8%), grade 1-2 arm lymphedema (8%) and grade 1-2 telangiectasia (6%).There was no significant difference in pain, radiation fibrosis, telangiectasia, arm lymphedema and dyspnea between the two groups. No patient experienced grade 3-4 toxicity in the two groups. Conclusions Our results indicate that concurrent bevacizumab with loco-regional RT provide acceptable early and late toxicities after one year in patients with non-metastatic breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-11.

The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as docosahexaenoic acid (DHA, 22:6n-3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n-3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n-3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.

Despite great improvements, a significant proportion of cancer patients still die, mainly because of the development of metastases. At this stage, current treatments still rely heavily on conventional chemotherapy for most cancers. The efficacy of chemotherapy is dose-dependent, which is limited by toxicity to non-tumor tissues, as a result of its poor tumor selectivity. To improve survival length and preserve quality of life, the challenge is to develop approaches aimed at increasing chemotherapy toxicity to tumor tissue while not affecting non-tumor tissues. Marine-derived lipids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have the potential to differentially sensitize tissues to chemotherapy. These lipids enhance the cytotoxicity of 15 anticancer drugs (antimetabolites, alkylating or intercalating agents, microtubule stabilizers, Abl tyrosine kinase inhibitor and arsenic trioxide) to a variety of cancer cell lines or tumors in animals, used as models for breast, prostate, colonic, lung, cervical, ovarian cancers, neuroblastomas, leukemia or lymphomas. However, DHA and EPA do not sensitize non-tumor tissues to anticancer drugs, which suggests that the effect of these lipids is tumor selective. Two phase II clinical trials already support these results, and randomized, phase III trials are ongoing. In this review, we discuss the double-faceted properties of these lipids, and then focus on their potential for transfer to the patient in the light of current therapeutic strategies. Should their beneficial effects be confirmed, the consequences could be considerable by opening up the prospect of systematic supplementation during cancer treatment, a significant shift in current cancer therapeutic paradigms.

Purpose This open label phase II study evaluated the safety and efficacy of vinflunine in patients with breast cancer previously treated with a vinorelbine-based regimen and who progressed during or within 6 months of completing this chemotherapy. Patients and methods Thirty eight patients received vinflunine 320 mg/m 2 once every 3 weeks. The primary efficacy endpoint was overall response rate (ORR). Secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety. Results ORR was 8.3% (95% CI: 1.75–22.4) and DCR was 75% (95% CI: 57.8–87.9). PFS was 4.0 months (95% CI: 2.5–6.1) and OS was 13.6 months (95% CI: 8.7–18.9). Toxicities not hampering dose intensity were as expected neutropenia (75.6% of patients), fatigue (44.7%), constipation (28.9%) and abdominal pain (26.3%). Conclusion Vinflunine demonstrated antitumour activity and can be safely administered in breast cancer patients refractory/resistant to vinorelbine.

BACKGROUND: ExteNET showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, significantly improves 2-year invasive disease-free survival after trastuzumab-based adjuvant therapy in women with HER2-positive breast cancer. We report updated efficacy outcomes from a protocol-defined 5-year follow-up sensitivity analysis and long-term toxicity findings. METHODS: In this ongoing randomised, double-blind, placebo-controlled, phase 3 trial, eligible women aged 18 years or older (≥20 years in Japan) with stage 1-3c (modified to stage 2-3c in February, 2010) operable breast cancer, who had completed neoadjuvant and adjuvant chemotherapy plus trastuzumab with no evidence of disease recurrence or metastatic disease at study entry. Patients who were eligible patients were randomly assigned (1:1) via permuted blocks stratified according to hormone receptor status (hormone receptor-positive vs hormone receptor-negative), nodal status (0 vs 1-3 vs or ≥4 positive nodes), and trastuzumab adjuvant regimen (given sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system, to receive 1 year of oral neratinib 240 mg/day or matching placebo. Treatment was given continuously for 1 year, unless disease recurrence or new breast cancer, intolerable adverse events, or consent withdrawal occurred. Patients, investigators, and trial funder were masked to treatment allocation. The predefined endpoint of the 5-year analysis was invasive disease-free survival, analysed by intention to treat. ExteNET is registered with ClinicalTrials.gov, number NCT00878709, and is closed to new participants. FINDINGS: Between July 9, 2009, and Oct 24, 2011, 2840 eligible women with early HER2-positive breast cancer were recruited from community-based and academic institutions in 40 countries and randomly assigned to receive neratinib (n=1420) or placebo (n=1420). After a median follow-up of 5·2 years (IQR 2·1-5·3), patients in the neratinib group had significantly fewer invasive disease-free survival events than those in the placebo group (116 vs 163 events; stratified hazard ratio 0·73, 95% CI 0·57-0·92, p=0·0083). The 5-year invasive disease-free survival was 90·2% (95% CI 88·3-91·8) in the neratinib group and 87·7% (85·7-89·4) in the placebo group. Without diarrhoea prophylaxis, the most common grade 3-4 adverse events in the neratinib group, compared with the placebo group, were diarrhoea (561 [40%] grade 3 and one [BACKGROUND: ExteNET showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, significantly improves 2-year invasive disease-free survival after trastuzumab-based adjuvant therapy in women with HER2-positive breast cancer. We report updated efficacy outcomes from a protocol-defined 5-year follow-up sensitivity analysis and long-term toxicity findings.METHODS: In this ongoing randomised, double-blind, placebo-controlled, phase 3 trial, eligible women aged 18 years or older (≥20 years in Japan) with stage 1-3c (modified to stage 2-3c in February, 2010) operable breast cancer, who had completed neoadjuvant and adjuvant chemotherapy plus trastuzumab with no evidence of disease recurrence or metastatic disease at study entry. Patients who were eligible patients were randomly assigned (1:1) via permuted blocks stratified according to hormone receptor status (hormone receptor-positive vs hormone receptor-negative), nodal status (0 vs 1-3 vs or ≥4 positive nodes), and trastuzumab adjuvant regimen (given sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system, to receive 1 year of oral neratinib 240 mg/day or matching placebo. Treatment was given continuously for 1 year, unless disease recurrence or new breast cancer, intolerable adverse events, or consent withdrawal occurred. Patients, investigators, and trial funder were masked to treatment allocation. The predefined endpoint of the 5-year analysis was invasive disease-free survival, analysed by intention to treat. ExteNET is registered with ClinicalTrials.gov, number NCT00878709, and is closed to new participants.FINDINGS: Between July 9, 2009, and Oct 24, 2011, 2840 eligible women with early HER2-positive breast cancer were recruited from community-based and academic institutions in 40 countries and randomly assigned to receive neratinib (n=1420) or placebo (n=1420). After a median follow-up of 5·2 years (IQR 2·1-5·3), patients in the neratinib group had significantly fewer invasive disease-free survival events than those in the placebo group (116 vs 163 events; stratified hazard ratio 0·73, 95% CI 0·57-0·92, p=0·0083). The 5-year invasive disease-free survival was 90·2% (95% CI 88·3-91·8) in the neratinib group and 87·7% (85·7-89·4) in the placebo group. Without diarrhoea prophylaxis, the most common grade 3-4 adverse events in the neratinib group, compared with the placebo group, were diarrhoea (561 [40%] grade 3 and one [INTERPRETATION: At the 5-year follow-up, 1 year of extended adjuvant therapy with neratinib, administered after chemotherapy and trastuzumab, significantly reduced the proportion of clinically relevant breast cancer relapses-ie, those that might lead to death, such as distant and locoregional relapses outside the preserved breast-without increasing the risk of long-term toxicity. An analysis of overall survival is planned after 248 events.FUNDING: Wyeth, Pfizer, and Puma Biotechnology.

// Maxime Gueguinou 1, 9 , Thomas Harnois 2, 9 , David Crottes 3 , Arnaud Uguen 6, 7 , Nadine Deliot 2, 9 , Audrey Gambade 1 , Aurelie Chantome 1, 9 , Jean Pierre Haelters 4, 9 , Paul Alain Jaffres 4, 9 , Marie Lise Jourdan 1, 10 , Gunther Weber 1 , Olivier Soriani 8 , Philippe Bougnoux 1, 9, 10 , Olivier Mignen 6, 9 , Nicolas Bourmeyster 2, 9 , Bruno Constantin 2, 9 , Thierry Lecomte 5, 9, 10 , Christophe Vandier 1, 9, * , Marie Potier-Cartereau 1, 9, * 1 INSERM UMR 1069, Universite de Tours, Tours, France 2 Equipe ERL 7368, CNRS, Universite de Poitiers, Poitiers, France 3 Department of Physiology, University of California, San Francisco, San Francisco, CA, USA 4 CNRS-UMR 6521-Universite de Brest, Brest, France 5 GICC-UMR 7292 Universite de Tours, Tours, France 6 INSERM-UMR 1078 Universite de Brest, Brest, France 7 CHRU Brest, Service d’Anatomie et Cytologie Pathologiques, Brest, France 8 CNRS UMR 7299, INSERM-UMR 1099, Universite de Nice Sophia-Antipolis, Nice, France 9 Ion Channels Network and Cancer-Canceropole Grand Ouest (IC-CGO), France 10 CHRU Tours, Tours, France * These authors have contributed equally to this work Correspondence to: Marie Potier-Cartereau, email: marie.potier-cartereau@univ-tours.fr Keywords: SOCE, Ohmline, lipid-raft channel complex, anti-EGFR mAbs, Akt signaling Received: September 24, 2015 Accepted: March 28, 2016 Published: April 18, 2016 ABSTRACT Background: Barely 10-20% of patients with metastatic colorectal cancer (mCRC) receive a clinical benefit from the use of anti-EGFR monoclonal antibodies (mAbs). We hypothesized that this could depends on their efficiency to reduce Store Operated Calcium Entry (SOCE) that are known to enhance cancer cells. Results: In the present study, we demonstrate that SOCE promotes migration of colon cancer cell following the formation of a lipid raft ion channel complex composed of TRPC1/Orai1 and SK3 channels. Formation of this complex is stimulated by the phosphorylation of the reticular protein STIM1 by EGF and activation of the Akt pathway. Our data show that, in a positive feedback loop SOCE activates both Akt pathway and SK3 channel activity which lead to SOCE amplification. This amplification occurs through the activation of Rac1/Calpain mediated by Akt. We also show that Anti-EGFR mAbs can modulate SOCE and cancer cell migration through the Akt pathway. Interestingly, the alkyl-lipid Ohmline, which we previously showed to be an inhibitor of SK3 channel, can dissociated the lipid raft ion channel complex through decreased phosphorylation of Akt and modulation of mAbs action. Conclusions: This study demonstrates that the inhibition of the SOCE-dependent colon cancer cell migration trough SK3/TRPC1/Orai1 channel complex by the alkyl-lipid Ohmline may be a novel strategy to modulate Anti-EGFR mAb action in mCRC.

International audience; Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.

Body weight loss during the course of cancer disease has been associated with poor prognosis. Beside cancer-associated cachexia, weight loss can also result from chemotherapy. This work explored whether a model of mammary tumors in female Sprague Dawley rats could be appropriate to study the effect of doxorubicin on body weight, described weight change in this model, and assessed the effect of DHA on weight during chemotherapy. After tumor induction, rats were randomly assigned to a control or a DHA-enriched diet, and treated with doxorubicin or placebo twice a week for 2.5 wk (n = 6 in each group). Body weight, food intake, and tumor growth were monitored. Neither the induction of tumors nor their initial development impaired body weight gain. No reduction in food intake was observed. Tumor growth was similar between groups from day 1 to day 11. Although doxorubicin induced body weight loss from day 4 compared to placebo (P< 0.01) in rats fed the control diet, it did not induce body weight loss in rats fed the DHA-enriched diet (P = 0.02), indicating that DHA had a protective effect. These results indicate that doxorubicin can induce body weight loss in this model and that a DHA-enriched diet can prevent this effect.

Developing approaches that will increase the selectivity of anticancer drugs remains a challenge. Docosahexaenoic acid (DHA) has the potential to increase tumor sensitivity to chemotherapy with no sensitization of normal tissues. This study was aimed at exploring the mechanism involved in this differential sensitization with a focus on oxidative stress, one of the main determinants involved in DHA enhancement of anthracycline-based chemotherapy.In a previously validated model of chemically induced mammary tumors in rats where supplemental DHA sensitized tumors to epirubicin, DHA level, lipid hydroperoxides (LPO), total antioxidant activity, glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities were measured in tumors, intestine, liver, and heart in rats treated with DHA+epirubicin and in control rats (palm oil, no chemotherapy).At baseline, the level of LPO was similar in tumors, liver, heart, and small intestine. The proportion of DHA was higher in non-tumor tissues compared to tumors (liver or heart, p0.0001; intestine, p = 0.01). DHA and epirubicin induced a significant increase in LPO in tumors (p = 0.03), but no increase was detected in liver, heart, or intestine. The difference in LPO production between these tissues was associated with differences in their antioxidant defenses. In tumors, the adjustment of GPx activity was possible but limited, and no adjustment of their total antioxidant and SOD activities was observed compared to other tissues.Supplementing DHA during an anthracycline-based chemotherapy induced a selective increase of LPO in tumors. This selectivity might result from a differential handling of oxidative stress between tumor and non-tumor tissues.

Summary Background Treatments with survival benefit are greatly needed for women with heavily pretreated metastatic breast cancer. Eribulin mesilate is a non-taxane microtubule dynamics inhibitor with a novel mode of action. We aimed to compare overall survival of heavily pretreated patients receiving eribulin versus currently available treatments. Methods In this phase 3 open-label study, women with locally recurrent or metastatic breast cancer were randomly allocated (2:1) to eribulin mesilate (1·4 mg/m 2 administered intravenously during 2–5 min on days 1 and 8 of a 21-day cycle) or treatment of physician's choice (TPC). Patients had received between two and five previous chemotherapy regimens (two or more for advanced disease), including an anthracycline and a taxane, unless contraindicated. Randomisation was stratified by geographical region, previous capecitabine treatment, and human epidermal growth factor receptor 2 status. Patients and investigators were not masked to treatment allocation. The primary endpoint was overall survival in the intention-to-treat population. This study is registered at ClinicalTrials.gov, number NCT00388726. Findings 762 women were randomly allocated to treatment groups (508 eribulin, 254 TPC). Overall survival was significantly improved in women assigned to eribulin (median 13·1 months, 95% CI 11·8–14·3) compared with TPC (10·6 months, 9·3–12·5; hazard ratio 0·81, 95% CI 0·66–0·99; p=0·041). The most common adverse events in both groups were asthenia or fatigue (270 [54%] of 503 patients on eribulin and 98 [40%] of 247 patients on TPC at all grades) and neutropenia (260 [52%] patients receiving eribulin and 73 [30%] of those on TPC at all grades). Peripheral neuropathy was the most common adverse event leading to discontinuation from eribulin, occurring in 24 (5%) of 503 patients. Interpretation Eribulin showed a significant and clinically meaningful improvement in overall survival compared with TPC in women with heavily pretreated metastatic breast cancer. This finding challenges the notion that improved overall survival is an unrealistic expectation during evaluation of new anticancer therapies in the refractory setting. Funding Eisai.

Undernutrition is frequently seen in cancer and is considered as a bad prognostic factor. The mechanisms involved in this complex syndrome are not fully understood. Systemic inflammation plays an important role in the occurrence and/or development of cancer-associated undernutrition. Although it is essential to counteract cancer-associated undernutrition, modalities for nutritional treatment are still discussed. It has been shown that nutritional supplementation with n-3 polyunsaturated fatty acid could be beneficial in patients.

BACKGROUND AND PURPOSE The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine) is an ether-linked phospholipid with promising anti-cancer properties but some side effects that preclude its full clinical therapeutic exploitation. We hypothesized that this lipid could interact with plasma membrane ion channels and modulate their function. EXPERIMENTAL APPROACH Using cell migration-proliferation assays, patch clamp, spectrofluorimetry and 125I-Apamin binding experiments, we studied the effects of edelfosine on the migration of breast cancer MDA-MB-435s cells, mediated by the small conductance Ca2+-activated K+ channel, SK3/KCa2.3. KEY RESULTS Edelfosine (1 µM) caused plasma membrane depolarization by substantially inhibiting activity of SK3/KCa2.3 channels, which we had previously demonstrated to play an important role in cancer cell migration. Edelfosine did not inhibit 125I-Apamin binding to this SKCa channel; rather, it reduced the calcium sensitivity of SK3/KCa2.3 channel and dramatically decreased intracellular Ca2+ concentration, probably by insertion in the plasma membrane, as suggested by proteinase K experiments. Edelfosine reduced cell migration to the same extent as known SKCa channel blockers. In contrast, K+ channel openers prevented edelfosine-induced anti-migratory effects. SK3 protein knockdown decreased cell migration and totally abolished the effect of edelfosine on MDA-MB-435s cell migration. In contrast, transient expression of SK3/KCa2.3 protein in a SK3/KCa2.3-deficient cell line increased cell migration and made these cells responsive to edelfosine. CONCLUSIONS AND IMPLICATIONS Our data clearly establish edelfosine as an inhibitor of cancer cell migration by acting on SK3/KCa2.3 channels and provide insights into the future development of a new class of migration-targeted, anti-cancer agents.

International audience; The aim of this study was to evaluate the effects of a fish oil (FO) diet (rich in long chain, n-3, polyunsaturated fatty acid) on cancer cachexia symptoms in rats. To this end, peritoneal carcinosis (PC) was generated by an intraperitoneal injection of cancer cells in BDIX rats fed FO or standard (Std) diets. Food intake and body weight were recorded throughout the study until sacrifice. PC rats were sacrificed when food intake was significantly and severely reduced. Fat and skeletal muscles masses were weighed and serum inflammatory cytokines concentration measured at sacrifice. Occurrence of anorexia in PC rats was delayed in the FO diet group (median time was multiplied by 2.5) in comparison with Std diet. At the time of sacrifice, PC rats displayed a lower body weight gain as well as lower muscle and fat masses than pair-fed rats, suggesting the presence of a hypermetabolism state. Serum TNF-alpha was significantly increased in PC rats compared with controls rats. There was no effect of FO diet on tissue mass (skeletal muscle and fat) or on TNF-alpha concentration. In conclusion, FO diet delays the appearance of anorexia induced by PC in rats.

The important factor determining breast cancer outcome is tumor cell sensitivity to cytotoxic agents. Most anticancer drugs lack intrinsic anti-tumor selectivity, provoking adverse effects in healthy nontumor tissues. As a result, drugs cannot always be delivered to the tumor at doses sufficient to induce tumor shrinkage, increasing the risk of the development of cellular drug resistance by tumor cells. Two main approaches are currently being investigated to reduce the toxicity in nontumor tissue by using a prodrug locally delivered through biochemical activation in the tumor environment, and for specifically increasing the sensitivity of tumors to anticancer drugs by modifying the tumor tissue through dietary supplementation. Both approaches have their own limitations that would be alleviated by combining them in order to better treat breast cancers. Specifically, the potential benefit of bringing together a glucuronide prodrug with dietary supplementation using polyunsaturated fatty acids, and in partic...

BACKGROUND: Breast cancer becomes lethal when visceral metastases develop. At this stage, anti-cancer treatments aim at relieving symptoms and delaying death without resulting in additional toxicity. On the basis of their differential anti-oxidant defence level, tumour cells can be made more sensitive to chemotherapy than non-tumour cells when membrane lipids are enriched with docosahexaenoic acid (DHA), a peroxidisable and oxidative-stress-inducing lipid of marine origin. METHODS: This open-label single-arm phase II study evaluated the safety and efficacy (response rate), as primary end points, of the addition of 1.8g DHA daily to an anthracycline-based chemotherapy (FEC) regimen in breast cancer patients (n ¼25) with rapidly progressing visceral metastases. The secondary end points were time to progression (TTP) and overall survival (OS). RESULTS: The objective response rate was 44%. With a mean follow-up time of 31 months (range 2–96 months), the median TTP was 6 months. Median OS was 22 months and reached 34 months in the sub-population of patients (n ¼12) with the highest plasma DHA incorporation. The most common grade 3 or 4 toxicity was neutropaenia (80%). CONCLUSION: DHA during chemotherapy was devoid of adverse side effects and can improve the outcome of chemotherapy when highly incorporated. DHA has a potential to specifically chemosensitise tumours.

Purpose: It has recently been shown that it is possible to improve the prediction of carboplatin clearance by adding plasma cystatin C level (cysC), an endogenous marker of glomerular filtration rate, to the other patient characteristics routinely used for carboplatin individual dosing, namely serum creatinine (Scr), actual body weight (ABW), age, and sex. This multicenter pharmacokinetic study was done to evaluate prospectively the benefit of using cysC for carboplatin individual dosing. Experimental Design: The 357 patients included in the study were receiving carboplatin as part of established protocols. A population pharmacokinetic analysis was done using NONMEM program. Seven covariates studied were as follows: Scr, cysC, age, sex, ABW, ideal body weight, and lean body mass. Results: The best covariate equation was as follows: carboplatin clearance (mL/min) = 117.8. (Scr/75)−0.450. (cysC/1,00)−0.385. (ABW/65)+0.504. (age/56)−0.366. 0.847sex, with Scr in μmol/L, cysC in mg/L, ABW in kilograms, age in years, and sex = 0 for male. Using an alternative weight descriptor (ideal body weight or lean body mass) did not improve the prediction. This final covariate model was validated by bootstrap analysis. The bias (mean percentage error) and imprecision (mean absolute percentage error) were +1% and 15%, respectively, on the total population, and were of a similar magnitude in each of the three subgroups of patients defined according to their body mass index. Conclusion: For the first time, a unique formula is proposed for carboplatin individual dosing to patients, which is shown to be equally valid for underweight, normal weight, and obese patients.

5-Fluorouracil (5-FU) is widely administered as a continuous infusion to treat gastrointestinal tract or head and neck cancers, and as bolus injections in breast cancer chemotherapy regimens. Grade 3–4 toxicity occurs in about 30% of patients receiving 5-FU as a continuous infusion, and proves lethal in 0.5% of these patients [1]. 5-FU-related toxicity has mostly been reported with intravenous administration [1–3]. However, some cases of severe toxicity, including deaths, have been described after oral administration of 5-FU derivatives, such as capecitabine (Xeloda®) [4, 5]. A polymorphism of the dihydropyrimidine dehydrogenase (DPD) gene has been identified as a frequent cause of such toxicity [6, 7]. DPD catalyses the rate-limiting step of fluoropyrimidine catabolism. Partial or total DPD deficiency therefore leads to substantial overexposure in patients treated with the standard dose, exacerbating drug toxicity. This raises questions about possible screening for DPD deficiency before the administration of fluoropyrimidine drugs, including their oral forms. Patients found to have a deficiency on screening before treatment or following signs of toxicity during a previous course are usually given alternative treatments based on nonfluoropyrimidine compounds. However, 5-FU is highly active and its use may be essential in patients who fail to respond to other treatments. We report the case of a patient with DPD deficiency detected due to severe toxicity during capecitabine treatment, who has since received a continuous infusion of 5-FU at almost the standard dose with no significant signs of toxicity.

International audience; OBJECTIVE: To evaluate the feasibility of in vivo measurement of the fatty acid (FA) composition of breast adipose tissue by MRS on a clinical platform. MATERIAL AND METHODS: MRS experiments were performed at 3 T, using a STEAM sequence, on 25 patients diagnosed with breast cancer. MR spectra, acquired on healthy breast tissue, were analysed with the LCModel. RESULTS: The measured values of the saturated fatty acid (SFA), mono-unsaturated fatty acid (MUFA) and poly-unsaturated fatty acid (PUFA) fractions were 23.8 ± 7.1 %, 55.4 ± 6.8 % and 20.8 ± 4.4 %, respectively. The values of SFA, MUFA and PUFA observed in the current study are in the same range as those found in two previous studies performed at 7 T. CONCLUSION: The results of the current study show that it is possible to quantify the fatty acid composition of breast tissue in vivo in a clinical setting (3 T)

64 p.-6 fig., Synthetic alkyl lipids, such as the ether lipids edelfosine (1-O-octadecyl-2-O-methyl-rac- glycero-3-phosphocholine) and ohmline (1-O-hexadecyl-2-O-methyl-rac-glycero-3-β- lactose), are forming a class of antitumor agents that target cell membranes to induce apoptosis and to decrease cell migration/invasion, leading to the inhibition of tumor and metastasis development. In this review, we present the structure-activity relationship of edelfosine and ohmline, and we point out differences and similarities between these two amphiphilic compounds. We also discuss the mechanisms of action of these synthetic alkyl ether lipids (involving, among other structures and molecules, membrane domains, Fas/CD95 death receptor signaling, and ion channels), and highlight a key role for lipid rafts in the underlying process. The reorganization of lipid raft membrane domains induced by these alkyl lipids affects the function of death receptors and ion channels, thus leading to apoptosis and inhibition of cancer cell migration. The possible therapeutic use of these alkyl lipids and the clinical perspectives for these lipids in prevention or/and treatment of tumor development and metastasis are also discussed., This work was supported by the Agence Nationale de la Recherche N°ANR-08-EBIO-020-01, la Ligue Contre le Cancer, Region Centre (LIPIDS project of ARD2020-Biomédicaments), INSERM, CNRS, Cancéropôle Grand Ouest, the association “CANCEN”, Tours’ Hospital oncology association ACORT", grants from Spanish Ministerio de Economia y Competitividad (SAF2011-30518, SAF2014-59716-R, and RD12/0036/0065 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union), and European Community’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011- 256986, PANACREAS). Ana Bouchet holds a post-doc mobility grant from PRESTIGE-European FP7 / University of Tours.

The present work investigates the relationship between cancer cell chemosensitivity and subcellular distribution, molecular interaction, and metabolism of an anticancer drug. To get insights into this relationship, we took advantage of the differential sensitivity of two breast cancer cell lines, MDA-MB-231 and MCF-7, to anthracyclines, along with the property of docosahexaenoic acid (DHA, 22:6n-3), to differentially enhance their cytotoxic activity. The fluorescent drug mitoxantrone (MTX) was used because of the possibility to study its subcellular accumulation by confocal spectral imaging (CSI). The use of CSI allowed us to obtain semiquantitative maps of four intracellular species: nuclear MTX bound to DNA, MTX oxidative metabolite in endoplasmic reticulum, cytosolic MTX, and finally, MTX in a low polarity environment characteristic of membranes. MDA-MB-231 cells were found to be more sensitive to MTX (IC50 = 18 nM) than MCF-7 cells (IC50 = 196 nM). According to fluorescence levels, the nuclear and cytosolic MTX content was higher in MCF-7 than in MDA-MB-231 cells, indicating that mechanisms other than nuclear MTX accumulation account for chemosensitivity. In the cytosol, the relative proportion of oxidized MTX was higher in MDA-MB-231 (60%) than in MCF-7 (7%) cells. DHA sensitized MDA-MB-231 (approximately 4-fold) but not MCF-7 cells to MTX and increased MTX accumulation by 1.5-fold in MDA-MB-231 cells only. The DHA-stimulated accumulation of MTX was attributed mainly to the oxidative metabolite. Antioxidant N-acetyl-L-cysteine inhibited the DHA effect on both metabolite accumulation and cell sensitization to MTX. We conclude that drug metabolism and compartmentalization are associated with cell chemosensitization, and the related cytotoxicity mechanisms may involve oxidative stress.

Peroxisome proliferator-activated receptor β (PPARβ) and NaV1.5 voltage-gated sodium channels have independently been shown to regulate human breast cancer cell invasiveness. The n-3 polyunsaturated docosahexaenoic acid (DHA, 22:6n-3), a natural ligand of PPAR, is effective in increasing survival and chemotherapy efficacy in breast cancer patient with metastasis. DHA reduces breast cancer cell invasiveness and it also inhibits PPARβ expression. We have shown previously that NaV1.5 promotes MDA-MB-231 breast cancer cells invasiveness by potentiating the activity of Na(+)/H(+) exchanger type 1 (NHE-1), the major regulator of H(+) efflux in these cells. We report here that DHA inhibited NaV1.5 current and NHE-1 activity in human breast cancer cells, and in turn reduced NaV1.5-dependent cancer cell invasiveness. For the first time, we show that antagonizing PPARβ, or inhibiting its expression, reduced NaV1.5 mRNA and protein expression and NaV1.5 current, as well as NHE-1 activity and cell invasiveness. Consistent with these results, the DHA-induced reduction of both NaV1.5 expression and NHE-1 activity was abolished in cancer cells knocked-down for the expression of PPARβ (shPPARβ). This demonstrates a direct link between the inhibition of PPARβ expression and the inhibition of Nav1.5/NHE-1 activities and breast cancer cell invasiveness. This study provides new mechanistic data advocating for the use of natural fatty acids such as DHA to block the development of breast cancer metastases.

A phase II study was conducted to evaluate the safety and efficacy of an oxaliplatin (OXA)/vinorelbine (VNB) combination in metastatic breast cancer (MBC) patients pre-treated with anthracyclines and taxanes. Patients received OXA at 130 mg/m 2 (2-h i.v.), day 1, and VNB days 1 and 8 at 24-26 mg/m 2 repeated every 3 weeks. Forty-two patients (median age 54; 64% with liver metastasis, 67% taxane resistant/refractory and 38% anthracycline resistant/refractory) were treated. A median of 4 cycles of treatment was given per patient, with 31% receiving 6 or more. Eleven partial responses and 16 patients with stable disease (five lasting more than 4 months) in 41 eligible patients were seen, for an overall response rate of 26.8% (95% confidence interval 14.2-42.9). Median follow-up was 15.9 months (7.2-30.6), median time to progression was 3.4 months and estimated overall survival was 12.7 months (20 events). Thirty-three patients experienced (National Cancer Institute Common Toxicity Criteria version 2) grade 3-4 neutropenia (one case of febrile neutropenia) and three patients had severe constipation requiring hospitalization. Nine patients developed grade 3 OXA-specific neurotoxicity. There were no treatment-related deaths. We conclude that OXA 130 mg/m 2 (day 1) and VNB 24 mg/m 2 (day 1 and 8) combination given every 3 weeks is effective with a good safety profile in MBC patients previously treated with anthracyclines and taxanes.

Prognostic factors are used to help clinical decision-making in selecting the appropriate treatment for individual patients. The purpose of this retrospective study was to identify one or more factors associated with overall survival (OS) and disease-free survival (DFS), in 710 patients with operable breast cancer, subjected to neoadjuvant chemotherapy followed by surgery, radiotherapy and adjuvant treatments. At a median follow-up of 7.6 years, univariate analysis showed that pathological complete response (pCR) was significantly related to survival (p < 0.003), as well as accepted prognostic factors, as SBR and MSBR grades, hormonal receptors or node involvement at surgery, who remained significant in our study (p < 0.001). The revised Nottingham prognostic index (NPI) and related indices (BGI, MNPI and MBGI) were also significantly associated to survival (p < 0.003). In multivariate analysis, node involvement and MSBR grade remained prognostic factors for OS and DFS (p < 0.0003 and p < 0.02, respectively). The MNPI and pCR were significantly related with OS (p = 0.04) and pts with hormonal receptor-positive tumours had a better DFS than others (p = 0.004). Among all clinical and pathological parameters, axillary dissection after neoadjuvant chemotherapy is still important to determine node involvement, a major prognostic factor. Moreover, MSBR grade seemed to be more accurate and predictive of long-term outcome than the standard SBR grade. It is concluded that, outside any other 'biological' factor, residual disease in breast and nodes must be strongly considered after an induction chemotherapy so as to choose adjuvant treatment for the individual patient.

Polyunsaturated fatty acids have been reported to enhance the cytotoxic activity of several anticancer drugs. In the present study, we observed that doxorubicin chemosensitization of breast cancer cell lines by docosahexaenoic acid (DHA, a long-chain omega-3 polyunsaturated fatty acid) was cell-line selective, affecting MDA-MB-231 and MCF-7 dox (a doxorubicin-resistant cell line) but not the parental MCF-7 cell line. DHA supplementation led to an increase in membrane phospholipid DHA level, but did not induce changes in intracellular [(14)C]doxorubicin accumulation. In MDA-MB-231, doxorubicin efficacy enhancement by DHA was linked to an increase in malondialdehyde level, a final product of lipid peroxidation. DHA elicited by itself a 3.7-fold malondialdehyde level increase, additive to that induced by doxorubicin. Addition of doxorubicin to DHA further increased the glutathione level, indicative of the generation of an oxidative stress. In contrast to MDA-MB-231, doxorubicin did not increase the malondialdehyde level in MCF-7, although DHA induced lipid peroxidation. Therefore in MCF-7, lipid peroxidation induced by DHA itself was not sufficient to trigger an oxidative stress and to subsequently increase sensitivity to doxorubicin. These data indicate that the differential effect of DHA among cells on drug toxicity results from a differential oxidative response to doxorubicin. Chemosensitization through fatty acids appears as a new promising adjuvant therapeutic paradigm, since omega-3 fatty acids are physiological molecules found in food and are nontoxic in vivo.

Les derives diene-conjugues de l’acide linoleique correspondent a une famille d’isomeres geometriques et positionnels de l’acide linoleique (isomeres diene-conjugues) collectivement denommes CLA (pour conjugated linoleic acid). Ce groupe d’acides gras, present dans les aliments d’origine animale comme la viande de ruminants ou dans certains produits laitiers [1], est egalement genere au cours de la cuisson [2], ainsi que sous l’action de la flore bacterienne intestinale [3]. L’isomere principal est le 9-cis,11-trans CLA. Cet isomere resulte aussi de la conversion endogene de l’acide vaccenique trans-11 18 :1, egalement present dans le lait. Les derives conjugues de l’acide linoleique (CLA) semblent avoir des proprietes inhibitrices de la cancerogenese, notamment mammaire. L’utilisation de differentes especes animales (souris, rates, hamsters), de diverses lignees cellulaires cancereuses, et de plusieurs modeles de cancerogenese (cancers chimio-induits avec des cancerigenes directs ou indirects, tumeurs transplantees) ont permis de confirmer le role protecteur des CLA en cancerogenese pour differents sites : peau [2], estomac [4], pancreas [5], foie [6], poumon [7], glandes mammaires [8-11] et colique [11-15]. Il a ete rapporte un effet inhibiteur des CLA aux differentes phases : initiation [9, 16, 17], promotion et croissance tumorales [8, 16], formation de metastases [18, 19]. Dans le modele de tumeurs mammaires chimio-induites, des rates prepuberes dont le regime etait supplemente en CLA avant l’initiation par un agent cancerigene (dimethylbenz(a)anthracene ou N-nitroso-N-methyluree), ont developpe significativement moins de tumeurs que les rates temoins (47 %) [9, 20], suggerant que la presence de CLA dans les tissus mammaires les rendraient moins sensibles a l’initiation ulterieure par un agent cancerigene. De meme, si la supplementation en CLA etait realisee apres l’initiation par un agent cancerigene pendant 20 semaines, les rates developpent significativement moins de tumeurs que les rates du lot temoin (32 a 60 % de tumeurs en moins) [8, 9]. Ces effets inhibiteurs de la promotion tumorale sont independants de la nature de l’agent cancerigene utilise ou des lipides (nature et quantite) administres dans le regime [21, 22]. L’apport de CLA dans la diete sous la forme d’acides gras libres semble avoir un effet similaire a celui des

The purpose of the study was to evaluate the efficacy and safety of docetaxel plus epirubicin (ET) and of 5-fluorouracil plus epirubicin and cyclophosphamide (FEC) as first-line chemotherapy for metastatic breast cancer. A total of 142 patients (intent-to-treat (ITT)) with at least one measurable lesion were randomised to receive docetaxel 75 mg m(-2) plus epirubicin 75 mg m(-2) or 5-fluorouracil 500 mg m(-2) plus epirubicin 75 mg m(-2) and cyclophosphamide 500 mg m(-2) intravenously once every 3 weeks for up to eight cycles. Prophylactic granulocyte-colony-stimulating factor was only permitted after the first cycle, if required. Per-protocol analysis (n=132) gave an overall response rate for ET of 63.1% (95% confidence interval (CI), 50-78%) and for FEC 34.3% (95% CI, 23-47%) after a median seven and six cycles, respectively. Intent-to-treat population (n=142) gave an overall response rate for ET of 59% (95% CI, 47-70%) and for FEC 32% (95% CI, 21-43%) after a median seven and six cycles, respectively. The median response duration for ET was 8.6 months (95% CI, 7.2-9.6 months) and for FEC 7.8 months (95% CI, 6.5-10.4 months). The median time to progression (ITT) for ET was 7.8 months (95% CI, 5.8-9.6 months) and for FEC 5.9 months (95% CI, 4.6-7.8 months). After a median follow-up of 23.8 months, median survival (ITT) for ET and FEC were 34 and 28 months, respectively. Nonhaematologic grade 3-4 toxicities were infrequent in both arms. Haematologic toxicity was more common with ET and febrile neutropenia was reported in 13 patients (18.6%) in the ET group. Two deaths in the ET group were possibly related to study treatment. In conclusion, both ET and FEC were associated with acceptable toxicity. ET is a highly active first-line therapy for metastatic breast cancer.

The purpose of the study was to determine the efficacy and safety of docetaxel plus continuous infusion of 5-fluorouracil (5-FU) in patients with metastatic breast cancer previously treated with anthracyclines. A total of 41 patients with histologically proven metastatic breast cancer and performance status 0-2, who had received at least one anthracycline-containing regimen, received docetaxel 85 mg m(-2) followed by continuous infusion of 5-FU 750 mg m(-2) day(-1) for 5 days every 3 weeks for up to eight cycles. All patients received corticosteroid premedication, but there was no prophylactic colony-stimulating factor support. The most frequent metastatic sites were the liver (61%), bone (29%), and lung (29%). All 41 patients were assessable for toxicity and 30 were eligible and assessable for efficacy. The objective response rate was 70.0% (95% CI: 53.6-86.4%) for the per protocol group and 53.7% (95% CI: 38.4-68.9%) for the intent-to-treat (ITT) population. For the ITT population, median duration of response was 8.4 months (95% CI: 6.7-12.2 months), median time to progression was 6.7 months (95% CI 5.5-8.6 months), and median survival was 17 months (95% CI: 12.3-not recorded months). Grade 3/4 neutropenia occurred in 54% of patients, with febrile neutropenia in 24% of patients and 5% of cycles, but infections were rare. Stomatitis was frequent, grade 3 in 24% of patients and grade 4 in one patient (2%), but manageable. Diarrhoea was rare, grade 3 in 7% of patients and 1% of cycles. Other grade 3/4 nonhaematological toxicities were infrequent. In conclusion, this docetaxel/5-FU regimen is highly active and well tolerated in patients with anthracycline-pretreated metastatic breast cancer. The efficacy is particularly promising, as one-third of patients were either second-line and/or anthracycline-resistant/refractory.

This study was designed to evaluate high-frequency power Doppler (PDS) and to quantify treatment-induced changes in an experimental autochthonous mammary tumour model in rats. A total of 13 rats with N-methyl, N-nitroso urea-induced mammary tumours were split into three courses; 6 rats were treated with epirubicin, 3 received a placebo injection and 4 had irradiation of their tumour with a direct electron beam using a single dose of 18 Gy. In all groups, treatment began when the tumour area reached at least 1 cm(2) and was preceded by the first power Doppler sonography study of the tumour (Echo #1). Echo #2 was carried out in the middle of the placebo or epirubicin treatment (after 3 weeks) or 7 days after irradiation in the irradiated group. Echo #3 was carried out at the end of placebo or epirubicin treatment or 28 days after irradiation. Then colour pixel density (CPD) and vascularity index (VI) were quantitated. Intraobserver and interobserver variability of the CPD and VI quantitation was low (r = 0.99 and 0.97, respectively, for intraobserver and interobserver variability of the CPD values). The monitoring of CPD and VI showed an increase with time during the observation period. No increase in CPD or VI was observed in treated mammary tumours (p < 0.01). Power Doppler sonography quantitation of angiogenesis is reproducible, noninvasive and feasible in this in vivo breast cancer model. The monitoring of angiogenesis according to different treatments is feasible in real-time. Further studies are needed to investigate the predictive value of CPD and VI on sensitivity of mammary tumours to anticancer treatment.

The aim of this study, using a Fleming single-stage design, was to explore the efficacy and safety of Taxotere® 100 mg m−2 docetaxel and FEC 75 cyclophosphamide 500 mg m−2, fluorouracil 500 mg m−2 and epirubicin 75 mg m−2, in alternating and sequential schedules for the first-line treatment of metastatic breast cancer. One hundred and thirty-six women were randomly allocated, to one of three treatment regimens: DTX 100 plus FEC 75, alternated for eight courses (ALT); four courses of DTX 100 followed by four courses of FEC 75 (SEQ T); or four courses of FEC 75 followed by four courses of DTX 100 (SEQ F). One hundred and thirty-one women were evaluable for tumour response. Although the treatment outcome was equivalent in the two sequential arms and the alternating regimen (P=0.110, not significant), the response rate was less encouraging in the SEQ F arm (52.3%) than in the other two arms (71.1% for ALT and 70.5% for SEQ T), in which docetaxel was administered first. Time to progression was similar in the ALT, SEQ T and SEQ F arms (9.5, 9.3 and 10.4 months respectively). Grade 3–4 neutropenia was observed in nearly all patients; febrile neutropenia occurred in 9% (ALT), 16% (SEQ T) and 2% (SEQ F) of patients. Few patients (⩽9%) developed grade 3–4 non-haematological toxicities. Relative dose intensity was 97–99% for all regimens. All treatment regimens were active and well tolerated. British Journal of Cancer (2002) 86, 692–697. DOI: 10.1038/sj/bjc/6600165 www.bjcancer.com © 2002 Cancer Research UK

Summary Background An effective and well tolerated treatment is needed for patients with early HER2-positive breast cancer who do not achieve a pathological complete response after neoadjuvant therapy. The AVATAXHER trial aimed to predict pathological complete response early with the use of PET and to investigate whether the addition of bevacizumab could improve the proportion of patients achieving a pathological complete response in patients unlikely to respond to treatment. Methods AVATAXHER was a randomised, open-label, non-comparative, multicentre phase 2 study that enrolled women (≥18 years of age) with early-stage HER2-positive breast cancer from 26 oncology centres in France. Patients initially received two cycles of neoadjuvant docetaxel (100 mg/m 2 intravenously every 3 weeks) plus trastuzumab (8 mg/kg intravenously every 3 weeks then 6 mg/kg intravenously every 3 weeks for the second course). Before the first and second cycles, [ 18 F]-fluorodeoxyglucose (FDG) PET was done and the change in standardised uptake value was used to predict pathological complete response in each patient. Patients who were predicted to be responders on PET continued to receive standard therapy. Predicted non-responders were randomly assigned (2:1) to receive four cycles of docetaxel (100 mg/m 2 intravenously every 3 weeks) and trastuzumab (6 mg/kg intravenously every 3 weeks) plus bevacizumab (15 mg/kg intravenously every 3 weeks; group A) or continue on docetaxel plus trastuzumab alone (group B). Randomisation was open label and was done by an adaptive minimisation method. Although investigators and patients were aware of group assignment, the anatomo-pathologist in charge of centralised review of surgical samples and lymph nodes was masked to treatment assignment. The primary endpoint was centrally assessed pathological complete response according to the Chevallier classification. Efficacy analyses were done in the intention-to-treat population. Safety analyses in this Article were done on all patients who received at least one dose of treatment starting from cycle 3. Survival outcomes are not yet mature. This study is registered with ClinicalTrials.gov (NCT01142778) and EUDRACT (2009-013410-26). Findings Between May 19, 2010, and Oct 1, 2012, 152 patients were recruited for the study. Ten patients were subsequently excluded, leaving 142 patients in the intention-to-treat population. Of these 142 patients, 69 were predicted by [ 18 F]-FDG PET to be treatment responders after two cycles of treatment. The 73 predicted non-responders were randomly assigned to group A (n=48) and group B (n=25). Pathological complete responses were noted in 37 (53·6%, 95% CI 41·2–65·7) of the PET responders, 21 (43·8%, 29·5–58·8) of those in group A, and six (24·0%, 9·4–45·1) of those in group B. Incidences of grade 3–4 adverse events were similar in all three groups. The most common grade 3–4 adverse events were neutropenia (four in PET responders, five in group A, and three in group B), febrile neutropenia (one, three, and one, respectively), and myalgia (four, none, and one, respectively). Overall, 24 serious adverse events were reported in 15 patients (PET responders: nine events in four [6%] of 67 patients; group A: 14 events in ten [21%] of 47 patients; group B: one event in one [4%] of 25 patients). No deaths occurred during the study. Interpretation In patients with HER2-positive breast cancer, early PET assessment can help to identify non-responders to neoadjuvant docetaxel plus trastuzumab therapy. In these patients, the addition of bevacizumab can increase the proportion of patients achieving a pathological complete response. This potential new role for PET and the activity of bevacizumab in this setting need to be confirmed in larger phase 3 trials. Funding Roche France.

The authors, all in charge of the administration of one of the 7 French Cancéropôles, reply to the article authored by Audrey Vézian, and -provide an alternative and more supportive view of the initiatives -sponsored by these regional cancer research networks.

Potassium channels belong to the largest and the most diverse super-families of ion channels. Among them, Ca(2+)-activated K(+) channels (KCa) comprise many members. Based on their single channel conductance they are divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa; SK1, SK2 and SK3). Ca(2+) channels are divided into two main families, voltage gated/voltage dependent Ca(2+) channels and non-voltage gated/voltage independent Ca(2+) channels. Based on their electrophysiological and pharmacological properties and on the tissue where there are expressed, voltage gated Ca(2+) channels (Cav) are divided into 5 families: T-type, L-type, N-type, P/Q-type and R-type Ca(2+). Non-voltage gated Ca(2+) channels comprise the TRP (TRPC, TRPV, TRPM, TRPA, TRPP, TRPML and TRPN) and Orai (Orai1 to Orai3) families and their partners STIM (STIM1 to STIM2). A depolarization is needed to activate voltage-gated Ca(2+) channels while non-voltage gated Ca(2+) channels are activated by Ca(2+) depletion of the endoplasmic reticulum stores (SOCs) or by receptors (ROCs). These two Ca(2+) channel families also control constitutive Ca(2+) entries. For reducing the energy consumption and for the fine regulation of Ca(2+), KCa and Ca(2+) channels appear associated as complexes in excitable and non-excitable cells. Interestingly, there is now evidence that KCa-Ca(2+) channel complexes are also found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration and the capacity to develop metastases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Membrane lipid rafts are distinct plasma membrane nanodomains that are enriched with cholesterol, sphingolipids and gangliosides, with occasional presence of saturated fatty acids and phospholipids containing saturated acyl chains. It is well known that they organize receptors (such as Epithelial Growth Factor Receptor), ion channels and their downstream acting molecules to regulate intracellular signaling pathways. Among them are Ca2+ signaling pathways, which are modified in tumor cells and inhibited upon membrane raft disruption. In addition to protein components, lipids from rafts also contribute to the organization and function of Ca2+ signaling microdomains. This article aims to focus on the lipid raft KCa/ClCa/Ca2+ channel complexes that regulate Ca2+ and EGFR signaling in cancer cells, and discusses the potential modification of these complexes by lipids as a novel therapeutic approach in tumor development. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

1044 Background: Combination chemotherapy (CT) is among the standard treatment options in metastatic breast cancer (MBC), especially in patients (pts) with visceral metastases or need of rapid symp...

Experimental studies have indicated that n-3 fatty acids, including alpha-linolenic acid (18:3 n-3) and long-chain n-3 polyunsaturated fatty acids inhibit mammary tumor growth and metastasis. Earlier epidemiological studies have given inconclusive results about a potential protective effect of dietary n-3 polyunsaturated fatty acids on breast cancer risk, possibly because of methodological issues inherent to nutritional epidemiology. To evaluate the hypothesis that n-3 fatty acids protect against breast cancer, we examined the fatty acid composition in adipose tissue from 241 patients with invasive, nonmetastatic breast carcinoma and from 88 patients with benign breast disease, in a case-control study in Tours, central France. Fatty acid composition in breast adipose tissue was used as a qualitative biomarker of past dietary intake of fatty acids. Biopsies of adipose tissue were obtained at the time of surgery. Individual fatty acids were measured as a percentage of total fatty acids, using capillary gas chromatography. Unconditional logistic regression modeling was used to obtain odds ratio estimates while adjusting for age, height, menopausal status and body mass index. We found inverse associations between breast cancer-risk and n-3 fatty acid levels in breast adipose tissue. Women in the highest tertile of alpha-linolenic acid (18:3 n-3) had an odds ratio of 0.39 (95% confidence intervals [CI] = 0.19–0.78) compared to women in the lowest tertile (trend p = 0.01). In a similar way, women in the highest tertile of docosahexaenoic acid (22:6 n-3) had an odds ratio of 0.31 (95% CI = 0.13-0.75) compared to women in the lowest tertile (trend p = 0.016). Women in the highest tertile of the long-chain n-3/total n-6 ratio had an odds ratio of 0.33 (95% confidence interval = 0.17–0.66) compared to women in the lowest tertile (trend p = 0.0002). In conclusion, our data based on fatty acids levels in breast adipose tissue suggest a protective effect of n-3 fatty acids on breast cancer risk and support the hypothesis that the balance between n-3 and n-6 fatty acids plays a role in breast cancer. © 2001 Wiley-Liss, Inc.