Epirubicin Treatment Research Articles

Systolic versus diastolic cardiac function variables during epirubicin treatment for breast cancer

Anthracyclines are important in the treatment of numerous malignant diseases but the use is limited by a risk of heart failure (CHF). LVEF (left ventricular ejection fraction) measurements by radionuclide ventriculography with multiple gated acquisition (MUGA) is often used for cardiac monitoring. However, diastolic variables have been proposed as sensitive supplements. It was hypothesized that a change in diastolic filling variables measured by MUGA could identify individuals after epirubicin treatment (ET) in risk of developing heart failure. A retrospective analysis of registered raw data. Individuals completing high-dose ET for breast cancer were selected from a 2-year period. All had MUGA-scans performed prior to and after ET and were observed clinically for late development of CHF. Eleven of 34 individuals developed CHF. A significant LVEF-reduction was recorded after ET with only minor changes in diastolic parameters. Development of CHF was related to dose, entry-blood pressure and inversely to post-epirubicin LVEF. Risk of CHF was high if LVEF <50% (Hazard ratio 3.31). Epirubicin induces considerable decrease in LVEF and a high risk of CHF. The risk of CHF is significantly higher if LVEF is reduced after ET. Diastolic MUGA-variables seem to add little information to conventional measurements of LVEF.

P62 An unexpected evolution of a pelvic osteolysis and cognitive disorders in a man treated for a renal-cell carcinoma

In metastatic breast cancer, nab-paclitaxel has been shown to significantly increase progression-free survival compared with solvent-based paclitaxel. The GeparSepto (GBG 69) trial assessed whether weekly nab-paclitaxel could increase the proportion of patients achieving pathological complete response compared with weekly solvent-based paclitaxel, both followed by epirubicin plus cyclophosphamide as neoadjuvant treatment.In a phase 3 randomised trial, we enrolled patients with previously untreated unilateral or bilateral primary invasive breast cancer and randomly assigned them in a 1:1 ratio using dynamic allocation and Pocock minimisation by breast cancer subtype, Ki67 and SPARC expression. Patients were treated for 12 weeks with either intravenous nab-paclitaxel 150 mg/m2 (after study amendment, 125 mg/m2) on days 1, 8, and 15 for four 3-week cycles, or solvent-based intravenous paclitaxel 80 mg/m2 on days 1, 8, and 15 for four 3-week cycles. Taxane treatment was followed in both groups by intravenous epirubicin 90 mg/m2 plus intravenous cyclophosphamide 600 mg/m2 on day 1 for four 3-week cycles. Patients with HER2-positive tumours received concurrent trastuzumab 6 mg/kg (loading dose 8 mg/kg) and pertuzumab 420 mg (loading dose 840 mg) on day 1 of every 3-week cycle. Trastuzumab and pertuzumab were given every 3 weeks concomitantly with chemotherapy for all cycles. This report is the final analysis of the primary endpoint, pathological complete response (ypT0 ypN0), analysed for all patients who started treatment (modified intention to treat). We used a closed test procedure to test for non-inferiority, with the nab-paclitaxel group calculated as non-inferior to the solvent-based paclitaxel group if the lower 95% CI for the OR was above 0·858 (OR equivalent to pathological complete response [33%] minus a 10% non-inferiority margin [3·3%]; 29·7%). We planned to test for superiority only in case of a positive non-inferiority test, using an α of 0·05. Safety was assessed in all patients who received study drug. The trial is registered with ClinicalTrials.gov, number NCT01583426.Between July 30, 2012, and Dec 23, 2013, we randomly assigned 1229 women, of whom 1206 started treatment (606 with nab-paclitaxel and 600 with solvent-based paclitaxel). The nab-paclitaxel dose was reduced after enrolment of 464 participants to 125 mg/m2 due to increased treatment discontinuation and sensory neuropathy in this group. Pathological complete response occurred more frequently in the nab-paclitaxel group (233 [38%, 95% CI 35–42] patients) than in the solvent-based paclitaxel group (174 [29%, 25–33] patients; OR 1·53, 95% CI 1·20–1·95; unadjusted p=0·00065). The incidence of grade 3–4 anaemia (13 [2%] of 605 patients in the nab-paclitaxel group vs four [1%] of patients in the solvent-based paclitaxel group; p=0·048) and peripheral sensory neuropathy grade 3–4 (63 [10%] patients receiving any nab-paclitaxel dose; 31 [8%] of patients starting with 125 mg/m2 and 32 [15%] of patients starting with 150 mg/m2; vs 16 [3%] in the solvent-based paclitaxel group, p<0·001) was significantly higher for nab-paclitaxel than for solvent-based paclitaxel. Overall, 283 (23%) patients were noted to have at least one serious adverse event (based on study drug received), 156 (26%) in the nab-paclitaxel group and 127 (21%) in the solvent-based paclitaxel group (p=0·057). There were three deaths (during epirubicin plus cyclophosphamide treatment) in the nab-paclitaxel group (due to sepsis, diarrhoea, and accident unrelated to the trial) versus one in the solvent-based paclitaxel group (during paclitaxel treatment; cardiac failure).Substituting solvent-based paclitaxel with nab-paclitaxel significantly increases the proportion of patients achieving a pathological complete response rate after anthracycline-based chemotherapy. These results might lead to an exchange of the preferred taxane, solvent-based paclitaxel, for nab-paclitaxel in therapy for primary breast cancer.Celgene, Roche.

Cardioprotective effect of telmisartan in cancer patients treated with epirubicin

e20671 Background: We previously showed on 31 cancer patients (pts) that early cardiac abnormalities occurred at epirubicin (EPI) doses of 200 mg/m2 and persisted throughout subsequent EPI doses and even up to 18 months. Early contractility impairment, i.e. Strain rate (SR) reduction was detected by tissue doppler imaging (TDI) associated with high levels of inflammatory/oxidative stress markers. Renin-angiotensin system activation has been suggested to play an important role in the pathogenesis of Anthracycline-induced cardiotoxicity. Methods: A phase II placebo-controlled study was designed to investigate the possible role of telmisartan (an antagonist of angiotensine II type I receptor) in preventing both early preclinical and late myocardial damage induced by EPI. The correlation with changes of biochemical/inflammatory markers was also assessed. Planned sample size was 100 pts (50 pts per arm). Inclusion criteria: 18–70 y, histologically confirmed cancer, previously untreated and candidates for an EPI-based regimen; LVEF ≥55%; ECOG PS 0–2, no history of cardiac disease and previous mediastinal irradiation. Eligible pts were randomized to receive telmisartan 40 mg (1 tablet)/day or placebo starting 1 week before EPI until 6 months after the end of EPI administration. TDI as well as inflammatory/oxidative stress markers were assessed at baseline, 24 hours and 7 days at EPI doses of 100, 200, 300, and 400 mg/m2. Results: At December 2008 we enrolled 27 pts (M/F: 7/20, mean±SD age 58±14 years): 14 telmisartan and 13 placebo. 15 pts completed EPI treatment (8 telmisartan and 7 placebo). A significant reduction of SR peak was observed at 200mg/m2 of EPI in the placebo arm. Viceversa no significant TDI changes occurred in the treatment arm. Proinflammatory cytokines did not change in both arms whilst reactive oxygen species increased significantly in the placebo arm. Conclusions: The study is in progress. No significant financial relationships to disclose.

Uridine glucuronosyltransferase 2B7 pharmacogenetics predicts epirubicin clearance and myelosuppression

2504 Background: Epirubicin (EPI) is widely used to treat breast cancer. EPI is predominantly metabolized by uridine glucuronosyltransferase (UGT) 2B7 to inactive glucuronides. We previously showed that a UGT enhancer single nucleotide polymorphism (SNP) at position -161 T to C correlated with rates of morphine glucuronidation. Methods: We performed a prospective pharmacogenetic study of effects of this SNP on EPI metabolism in M0 breast cancer patients (PTS) receiving adjuvant or neoadjuvant FEC100 (5-fluorouracil 500 mg/m2, EPI 100 mg/m2 and cyclophosphamide 500 mg/m2) given every 3 wks. PTS with ALT and AST ≤ upper limit of normal (ULN), a total bilirubin ≤ ULN, and normal renal and cardiac function were eligible. EPI levels were drawn at approximately 1 and 24 hrs. Cycle 1 toxicities were assessed using NCIC CTG toxicity criteria. Results: 123 PTS entered this study, mean (range): age 51 (28 - 74), sex 122 F/ 1 M, baseline AST 24 U/L (13–66), ALT 22 U/L (5–90), bilirubin 8 μmol/L (2–26), creatinine 74 μmol/L (50 - 126). PTS were genotyped using Pyrosequencing; 26 PTS were TT homozygotes, 59 were CT heterozygotes, and 33 were CC homozygotes. 5 PTS could not be genotyped. A three compartment population pharmacokinetic model in NONMEM V 1.1 for EPI was used incorporating all PTS. The baseline objective function was 1817, and inclusion of genotype significantly improved the objective function to 1764; CC genotype PTS had decreased EPI clearance 88.9 L/hr compared to CT/TT genotype PTS 129 L/hr, p&lt;0.001. Rates of first cycle grade 3/4 leucopenia were 78% in CC PTS and 48% in CT/TT PTS; consistent with the pharmacokinetic analysis. Conclusions: A SNP in UGT 2B7 is common and appears to predicts EPI clearance and myelosuppresion in non-metastatic breast cancer PTS and may form the basis for a method to individualize EPI treatment. No significant financial relationships to disclose.

Side effects after docetaxel treatment in Taiwanese breast cancer patients with CYP3A4, CYP3A5, and ABCB1 gene polymorphisms

Background Breast cancer patients initiating TEC (including docetaxel, epirubicin, and cyclophosphamide) treatment were genotyped for CYP3A4, CYP3A5, and ABCB1 to identify variability factors of side effects for docetaxel. Methods The planned dose of docetaxel per course was formulated according to each patient's height and weight. Each participant had received TEC treatment for 6 consecutive cycles. The single nucleotide polymorphisms (SNPs) of CYP3A4⁎4 (352A > G), CYP3A4⁎5 (653C > G), and CYP3A4⁎18A (20070T > C) for the CYP3A4 gene, CYP3A5⁎3A (6986A > G) for the CYP3A5 gene, and − 41A > G, − 145C > G, 1236C > T, 2677G > T(A), and 3435C > T SNPs for the ABCB1 gene were determined by using the restriction fragment length polymorphism of polymerase chain reaction products and the restriction enzymes. Results Fifty-nine Taiwanese women (mean age, 46 y; range, 30–64 y) treated for breast cancer with TEC were recruited. We found that patients carrying the CYP3A5⁎1/⁎3 genotype demonstrated more side effects of fever, pleural effusion, and febrile neutropenia than those with the CYP3A5⁎3/⁎3 genotype ( p = 0.075, 0.077, and 0.030, respectively); moreover, patients with the ABCB1 2677G/G genotype also showed more side effects of fever and febrile neutropenia than those with other genotypes ( p = 0.024 and 0.027), In regard to ABCB1 3435C > T, patients with ABCB1 3435C/C tended to suffer leucopenia ( p = 0.057). Conclusions There could be correlations between certain side effects of docetaxel treatment and polymorphisms of these metabolic enzymes. Unfortunately, there is not so much evidence due to the small sample size of this study which restricts the statistical power.

Perturbation of DNA repair pathways by proteasome inhibitors corresponds to enhanced chemosensitivity of cells to DNA damage-inducing agents

PurposeBreast cancer treatment often employs DNA double-strand breaks (DSBs), such as that induced by irradiation or anticancer agents. Ubiquitination is required at the site of DNA damage and plays a crucial role in the DSB repair pathway. We investigated the effect of proteasome inhibitors on the pathway after exposure to chemotherapeutic agents and examined its correlation with cytotoxicity.MethodsCells were exposed for 1 h to DNA damage-inducing chemotherapeutic agents. After DNA damage, nuclear foci formation of conjugated ubiquitin (Ub-foci) and cell viability were examined in the absence or presence of proteasome inhibitors MG132 and epoxomicin.ResultsProteasome inhibitors trapped conjugated ubiquitin in the cytosol and blocked irinotecan (CPT-11)- and epirubicin-induced Ub-foci formation in MCF10A cells and HeLa cells, but not in MCF7 cells. MG132 sensitized MCF10A cells to CPT-11 and epirubicin treatment, demonstrating a synergistic effect. This synergistic effect is likely due to the failure to repair DNA, because a significant rise in unrepaired DNA damage was observed in the cells treated with MG132. On the other hand, no synergy was observed in MCF7 cells or when MG132 was combined with docetaxel.ConclusionsThe synergistic effect of proteasome inhibitors in combination with DNA damage-inducing agents warrants further investigating into its effectiveness in the treatment of breast cancer.

Epirubicin/Vinorelbine Adjuvant Chemotherapy in Young Women with Breast Cancer is Associated with Preservation of Menstrual Function

Aims To determine whether the epirubicin and vinorelbine regimen in the adjuvant (neoadjuvant) treatment of breast cancer has minimum adverse effects on menstrual function. Patients and methods Thirty-six premenopausal women with a median age of 32 (25–47) years received epirubicin and vinorelbine. Twenty-eight received only epirubicin and vinorelbine without any other neo/adjuvant chemotherapy agents. Amenorrhoea was defined as absence of periods 6 months after the completion of chemotherapy. The medical records of all patients were reviewed retrospectively. Results Twenty-six patients were assessable for effects of epirubicin and vinorelbine on menstruation. All the 26 patients resumed menstruation within 6 months of completing epirubicin and vinorelbine treatment. Epirubicin and vinorelbine was well tolerated. After a median follow-up of 38.5 (11–78) months, six (21%) patients had developed disease relapse and three (11 %) had died. The 6.5-year disease-free survival and overall survival probabilities were 77 and 86%, respectively. Conclusion Adjuvant (neoadjuvant) epirubicin and vinorelbine is an effective and well-tolerated regimen that is associated with the retention of menstrual function.

Dose-Finding Phase I and Pharmacokinetic Study of Capecitabine (Xeloda) in Combination with Epirubicin and Cyclophosphamide (CEX) in Patients with Inoperable or Metastatic Breast Cancer

Objective: The present study aimed to identify the recommended dose (RD) of capecitabine, epirubicin and cyclophosphamide (CEX) combination treatment for inoperable or metastatic breast cancer and to assess response rate, tolerability and pharmacokinetics in Japanese patients. Methods: Patients received 3-week cycles of fixed-dose intravenous cyclophosphamide (600 mg/m², day 1), intravenous epirubicin (75–100 mg/m², day 1) and oral capecitabine (628–900 mg/m² twice daily, days 1–14). Dose escalation/deescalation decisions for consecutive cohorts were made using the continual reassessment method (CRM). RD was defined as dose level closest to that causing dose-limiting toxicity (DLT) in 33% of patients. Results: Among the 17 patients enrolled, 3 experienced DLT (hand-foot syndrome, anorexia, mucositis; all grade 3) and 3 (18%) febrile neutropenia. Based on CRM calculation, mean DLT occurrence probabilities (90% confidence intervals in parentheses) for levels 2, 3 and 4 were 21% (8–42%), 35% (17–56%) and 56% (38–71%), respectively. Level 3 was thus chosen as the RD. Combination treatment did not affect pharmacokinetic parameters of capecitabine, epirubicin or their metabolites. Objective responses were achieved by 7 patients (41%), including partial responses in 4 of 6 patients (67%) receiving the RD. Conclusion: CEX represents a well-tolerated regimen, and RD for Japanese patients was determined to be capecitabine 900 mg/m² twice daily in combination with epirubicin 90 mg/m² and cyclophosphamide 600 mg/m² using the CRM.

Comparison of Three Different Chemotherapy Regimens Containing Epirubicin in Hormone-Refractory Prostate Cancer Patients

We compared three different chemotherapy regimens containing epirubicin in hormonerefractory prostate cancer (HRPC) patients. Sixty-nine patients with HRPC were randomized into three groups. The first group (22 patients) received 30 mg/m2/week i.v. epirubicin for 8 weeks. The second group (24 patients) received 30 mg/m2/week i.v. epirubicin for 8 weeks followed by monthly maintenance therapy for 4–6 months. The third group (23 patients) received oral estramustine phosphate (EMP) at a dose of 840 mg/day together with weekly and monthly maintenance epirubicin. The response rates, mean survival times, and toxicity were determined. Within the first 3 months, pain and performance scores were improved by at least one degree in all the groups. One patient in group two and three patients in group three had complete response. Partial response rates were 23% in group 1, 25% in group 2, and 17% in group 3. Stable disease rates were 41% in group 1, 33% in group 2, and 26% in group 3. The progression rates within the first 3 months were 36% in group 1, 38% in group 2, and 44% in group 3. None of the patients developed complications that were significant enough to terminate the treatment. Two patients in group 3 died of cardiotoxicity. The mean survival times were 10.1, 15.8, and 16.1 months in groups 1, 2, and 3, respectively. It was determined that weekly and maintenance epirubicin treatment protocol, and estramustine treatment protocol in addition to this treatment, was only meaningfully more effective against weekly epirubicin treatment in the statistical sense (0.01 < p < 0.05). However, due to the complications of EMP, which influence the quality of life, we believe that this was usable only when measures were adopted against these effects.

Epirubicin in the adjuvant treatment of splenic hemangiosarcoma in dogs: 59 cases (1997–2004)

To determine the efficacy and toxic effects of epirubicin for the adjuvant treatment of dogs with splenic hemangiosarcoma and identify prognostic factors. Retrospective case series. 59 client-owned dogs that underwent splenectomy for splenic hemangiosarcoma treated with or without epirubicin. Medical records were examined for signalment, clinical signs, diagnostic and surgical findings, and postoperative outcome. For dogs treated with epirubicin, dose numbers, intervals, and reductions and type and severity of toxic effects were recorded. Dogs were allotted to 2 groups: splenectomy alone and splenectomy with adjuvant epirubicin treatment. 18 dogs received epirubicin (30 mg/m(2)) every 3 weeks for up to 4 to 6 treatments. Forty-one dogs were treated with splenectomy alone. The overall median survival time was significantly longer in dogs treated with splenectomy and epirubicin (144 days), compared with splenectomy alone (86 days). Median survival time for dogs with stage I disease (345 days) was significantly longer than for dogs with either stage II (93 days) or III disease (68 days). Seven of 18 dogs treated with epirubicin were hospitalized for signs of adverse gastrointestinal effects. Inappetence, long duration of clinical signs, thrombocytopenia, neutrophilia, and high mitotic rate were negative prognostic factors. Epirubicin may be as efficacious as adjuvant doxorubicin-based protocols, but may result in a higher incidence of adverse gastrointestinal effects. Epirubicin should be considered as an alternative to doxorubicin in dogs with preexisting cardiac disease, as clinical epirubicin cardiotoxicity was not diagnosed in treated dogs.

18F]2-Fluoro-2-deoxy-D-glucose incorporation by AGS gastric adenocarcinoma cells in vitro during response to epirubicin, cisplatin and 5-fluorouracil

Decreased tumour [18F]2-fluoro-2-deoxy-D-glucose (18FDG) incorporation is related to response however its significance at the cell level in gastro-oesophageal cancer and how it relates to cell death is unknown. Here human gastric adenocarcinoma (AGS) cells were treated with lethal dose 10 and 50 (LD10 and LD50), determined by using the MTT assay, of the three drugs, epirubicin, 5-fluorouracil and cisplatin, commonly used in the treatment of patients with gastro-oesophageal cancer. 18FDG incorporation was determined after 48 and 72 h of treatment with each drug and related to drug-induced changes in glucose transport, hexokinase activity, cell cycle distribution and annexin V-PE binding (a measure of apoptosis). Treatment of cells for 48 and 72 h with LD50 doses of cisplatin resulted in reductions in 18FDG incorporation of 27 and 25% respectively and of 5-fluorouracil reduced 18FDG incorporation by 34 and 33% respectively: epirubicin treatment reduced incorporation by 30 and 69% respectively. Cells that had been treated for 72 h with each drug were incubated in drug-free media for a further 6 days to determine their ability to recover. Comparison of the ability to recover from the chemotherapy agent, with 18FDG incorporation before the recovery period allowed an assessment of the predictive ability of 18FDG incorporation. Cells treated with either 5-fluorouracil or cisplatin demonstrated recovery on removal of the drug. In contrast, cells treated with epirubicin did not recover corresponding with the greatest 72 h treatment decrease in 18FDG incorporation. In contrast to adherent cells treated with cisplatin or 5-fluorouracil, adherent epirubicin-treated cells also exhibited very high levels of apoptosis. Glucose transport was decreased after each treatment whilst hexokinase activity was only decreased after 72 h of treatment with each drug. There was no consistent relationship observed between 18FDG incorporation and cell cycle distribution. Our results show that at the tumour cell level in gastric tumour cells, decreased 18FDG incorporation and glucose transport, accompanies therapeutic growth inhibition. 18FDG incorporation is particularly diminished in cells exhibiting apoptosis.

Altered chemotherapeutic response of primary human breast cancer epithelial cells (HBCEC) and breast cancer cell lines

11506 Background: A novel technique to obtain individual primary cultures of human breast cancer biopsies was filed for patent (PCT/DE 2006/000608). The different individualized HBCEC (human breast cancer epithelial cell) cultures will be characterized and chemotherapeutic effects will be compared to established breast cancer cell lines. Methods: Primary HBCEC from 20 different breast cancer patients were characterized for epithelial cell and tumor markers by immunofluorescence and PCR. Following treatment with 1μM epirubicin for 1h up to 72h differences in protein expression patterns were compared to the similarly treated MCF-7 cell line by 2D gel electrophoresis. Differentially expressed protein spots were identified by mass spectrometry and confirmed by appropriate Western blot analysis. Results: Characterization of primary HBCEC revealed continuous mitosis and cell cycle progression for more than one year in culture with no significant contamination by fibroblasts or other cell types. Whereby HBCEC underwent cell death within 72h of epirubicin treatment analysis by 2D gel and subsequent protein identification by MALDI-TOF/TOF mass spectrometry exhibited a variety of differences compared to MCF-7 cells including HSP27 and prohibitin. Appropriate Western blots confirmed these differences and revealed altered expression levels for HSP27 and prohibitin in the course of epirubicin exposure in HBCEC and MCF-7 cells, respectively, suggesting altered signalling pathways in either primary breast cancer cells or the tumor cell line. Conclusions: Individualized primary HBCEC from various patients could provide a cellular platform beyond breast cancer cell lines, which eventually meet the requirements for an appropriate breast cancer testing system including the characterization of biomarkers and the identification of potential molecular targets. No significant financial relationships to disclose.

Supplementation with l-carnitine does not reduce the efficacy of epirubicin treatment in breast cancer cells

One of the cornerstones of therapy for invasive breast cancer includes the use of anthracyclines. Epirubicin, a stereoisomer of doxorubicin, is one of the commonly used anthracyclines. Anthracyclines while effective therapy for breast cancer, have their own unique toxicities, such as cardiomyopathy. l-Carnitine, a quarternary ammonium compound synthesized from methionine and lysine, is required for oxidative metabolism in mitochondria. Cardiac function is closely linked with oxidative metabolism whereby l-carnitine is an essential cofactor. A hypothesis is being investigated to determine if supplementation with carnitine in breast cancer patients treated with epirubicin will reduce the development of cardiac toxicity. We determined whether addition of l-carnitine altered the tumor cytotoxic effects of epirubicin using a number of in vitro cell viability assays in different breast cancer cell lines including BT549, MDA-MB-435, NCI-ADR-RES, MCF7 and T47D. Additionally we investigated the ability of cells to respond to l-carnitine following analysis of the expression of carnitine metabolic enzymes by RT-PCR. We determined that supplementation with l-carnitine had no effect on the ability of epirubicin to kill a variety of breast cancer cell lines. Additionally, no differences in the induction of apoptosis by epirubicin were observed. Furthermore, all cell lines examined expressed proteins required for carnitine uptake and use. Our data suggest that supplementation with l-carnitine does not impair the ability of epirubicin to kill breast cancer cells. These results suggest that supplementation with l-carnitine in patients undergoing epirubicin treatment could be safely used to reduce associated cardiotoxicities without fear that the efficacy of chemotherapy is jeopardized.

BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

The breast and ovarian tumor suppressor BRCA1 catalyzes untraditional polyubiquitin chains that could be a signal for processes other than proteolysis. However, despite intense investigations, the mechanisms regulated by the enzyme activity remain only partially understood. Here, we report that BRCA1-BARD1 mediates polyubiquitination of RPB8, a common subunit of RNA polymerases, in response to DNA damage. A proteomics screen identified RPB8 as a protein modified after epirubicin treatment in BRCA1-dependent manner. RPB8 interacted with BRCA1-BARD1 and was polyubiquitinated by BRCA1-BARD1 in vivo and in vitro. BRCA1-BARD1 did not destabilize RPB8 in vivo but rather caused an increase in the amount of soluble RPB8. Importantly, RPB8 was polyubiquitinated immediately after UV irradiation in a manner sensitive to BRCA1 knockdown by RNA interference. Substitution of five lysine residues of RPB8 with arginine residues abolished its ability to be ubiquitinated while preserving its polymerase activity. HeLa cell lines stably expressing this ubiquitin-resistant form of RPB8 exhibited UV hypersensitivity accompanied by up-regulated caspase activity. Our findings suggest that ubiquitination of a common subunit of RNA polymerases is a mechanism underlying BRCA1-dependent cell survival after DNA damage.

Does Granulocyte Colony-Stimulating Factor Worsen Anemia in Early Breast Cancer Patients Treated With Epirubicin and Cyclophosphamide?

We report on the effects of granulocyte colony-stimulating factor (G-CSF) on hemoglobin (Hb) value in early breast cancer patients receiving high-dose epirubicin and cyclophosphamide (EC) adjuvant treatment. Five hundred and six stage I or stage II female breast cancer patients were treated with E 120 mg/m2 and C 600 mg/m2 with or without G-CSF and randomly assigned to receive in a factorial 2 x 2 design: EC; EC + lonidamine; EC + G-CSF; EC + lonidamine + G-CSF. Five consecutive G-CSF schedules tested 100 randomly assigned patients each: (1) 480 microg subcutaneously on days 8 to 14; (2) 480 microg on days 8, 10, 12, 14; (3) 300 microg on days 8 to 14; (4) 300 microg on days 8, 10, 12, and 14; and (5) 300 microg on days 8 and 12. The mean Hb level of 246 patients receiving EC plus G-CSF was compared with that of 240 patients receiving EC alone. The data presented are derived from an exploratory hypothesis-generating analysis. The EC dose intensity did not statistically differ between the G-CSF and the control arm. From the third cycle onward, the mean Hb value resulted significantly lower in G-CSF arm compared with control at each time point of each cycle (P < .0001). No statistically significant difference in the mean Hb level was observed between schedule 5 and control. Of interest, from the second course onward, the mean Hb level tended to be lower in patients receiving seven or four G-CSF injections compared with those patients who received only two injections. Our data suggest that a G-CSF dose-related effect may play a role in worsening anemia in patients receiving adjuvant EC.

258 ORAL Utilisation of microarray technology to refine molecular classes and improve clinical management of breast cancer

In metastatic breast cancer, nab-paclitaxel has been shown to significantly increase progression-free survival compared with solvent-based paclitaxel. The GeparSepto (GBG 69) trial assessed whether weekly nab-paclitaxel could increase the proportion of patients achieving pathological complete response compared with weekly solvent-based paclitaxel, both followed by epirubicin plus cyclophosphamide as neoadjuvant treatment.In a phase 3 randomised trial, we enrolled patients with previously untreated unilateral or bilateral primary invasive breast cancer and randomly assigned them in a 1:1 ratio using dynamic allocation and Pocock minimisation by breast cancer subtype, Ki67 and SPARC expression. Patients were treated for 12 weeks with either intravenous nab-paclitaxel 150 mg/m2 (after study amendment, 125 mg/m2) on days 1, 8, and 15 for four 3-week cycles, or solvent-based intravenous paclitaxel 80 mg/m2 on days 1, 8, and 15 for four 3-week cycles. Taxane treatment was followed in both groups by intravenous epirubicin 90 mg/m2 plus intravenous cyclophosphamide 600 mg/m2 on day 1 for four 3-week cycles. Patients with HER2-positive tumours received concurrent trastuzumab 6 mg/kg (loading dose 8 mg/kg) and pertuzumab 420 mg (loading dose 840 mg) on day 1 of every 3-week cycle. Trastuzumab and pertuzumab were given every 3 weeks concomitantly with chemotherapy for all cycles. This report is the final analysis of the primary endpoint, pathological complete response (ypT0 ypN0), analysed for all patients who started treatment (modified intention to treat). We used a closed test procedure to test for non-inferiority, with the nab-paclitaxel group calculated as non-inferior to the solvent-based paclitaxel group if the lower 95% CI for the OR was above 0·858 (OR equivalent to pathological complete response [33%] minus a 10% non-inferiority margin [3·3%]; 29·7%). We planned to test for superiority only in case of a positive non-inferiority test, using an α of 0·05. Safety was assessed in all patients who received study drug. The trial is registered with ClinicalTrials.gov, number NCT01583426.Between July 30, 2012, and Dec 23, 2013, we randomly assigned 1229 women, of whom 1206 started treatment (606 with nab-paclitaxel and 600 with solvent-based paclitaxel). The nab-paclitaxel dose was reduced after enrolment of 464 participants to 125 mg/m2 due to increased treatment discontinuation and sensory neuropathy in this group. Pathological complete response occurred more frequently in the nab-paclitaxel group (233 [38%, 95% CI 35–42] patients) than in the solvent-based paclitaxel group (174 [29%, 25–33] patients; OR 1·53, 95% CI 1·20–1·95; unadjusted p=0·00065). The incidence of grade 3–4 anaemia (13 [2%] of 605 patients in the nab-paclitaxel group vs four [1%] of patients in the solvent-based paclitaxel group; p=0·048) and peripheral sensory neuropathy grade 3–4 (63 [10%] patients receiving any nab-paclitaxel dose; 31 [8%] of patients starting with 125 mg/m2 and 32 [15%] of patients starting with 150 mg/m2; vs 16 [3%] in the solvent-based paclitaxel group, p<0·001) was significantly higher for nab-paclitaxel than for solvent-based paclitaxel. Overall, 283 (23%) patients were noted to have at least one serious adverse event (based on study drug received), 156 (26%) in the nab-paclitaxel group and 127 (21%) in the solvent-based paclitaxel group (p=0·057). There were three deaths (during epirubicin plus cyclophosphamide treatment) in the nab-paclitaxel group (due to sepsis, diarrhoea, and accident unrelated to the trial) versus one in the solvent-based paclitaxel group (during paclitaxel treatment; cardiac failure).Substituting solvent-based paclitaxel with nab-paclitaxel significantly increases the proportion of patients achieving a pathological complete response rate after anthracycline-based chemotherapy. These results might lead to an exchange of the preferred taxane, solvent-based paclitaxel, for nab-paclitaxel in therapy for primary breast cancer.Celgene, Roche.

Outcome for breast cancer patients with congestive heart failure after epirubicin treatment

622 Background: Patients (pts) treated with Epirubicin (EPI) are at risk for development of cardiomyopathy and congestive heart failure (CHF). Only limited data are available for the outcome of these pts. Methods: In a retrospective study, the history of 1122 consecutive pts with metastatic breast cancer (BC) was retrieved. All were treated with EPI alone or EPI in combination with other cytostatics from November 1983 - November 2003. Pts with cardiac disease New York Heart Association (NYHA) Class II were included. Statistical analyses included Kaplans-Meiers plot and Cox multivariate analysis. Results: 116 pts (10.3%) developed CHF. Time from discontinuation of EPI to onset of CHF was 3 mths (range 0–58). The median total dose of EPI among pts with CHF was 933 mg/m2 (range 260–1433). The median age was 60 years (range 38 - 79). 25.9% of the pts had symptoms according to NYHA II, 31.9% to NYHA III, 33.6% to NYHA IV and for 8.6% the exact NYHA class was unknown. The pts were treated with a single drug or with a combination of digoxin (56%), diuretics (78%), and ACE inhibitors (53%). 42.2% of the pts had benefit of the treatment, for 25.9% the symptoms were unchanged, the symptoms progressed in 24.1%, and in 7.8% the outcome was unknown. The median follow-up for pts with CHF was 73 mths (range 0 - 124 +). The median survival after CHF was 5.9 mths (range 0 - 124 +). 9.5% of the pts is still alive, 23.3% died of CHF, 41.4% of BC, 16.4% of a combination of CHF and BC, and for 8.6% the cause of dead was unknown. The following factors became significant for survival in a univariate analysis: the BC status at onset of CHF and the pts NYHA classification. In a Cox multivariate analysis only BC status became significant, p-value= 0.007. Conclusions: The number of pts who developed CHF after treatment with EPI was substantial. Only pts BC status was predictive for the survival after the onset of CHF. No significant financial relationships to disclose.

In vivo quantitation of tumour vascularisation assessed by Doppler sonography in rat mammary tumours

This study was designed to evaluate high-frequency power Doppler (PDS) and to quantify treatment-induced changes in an experimental autochtonous 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. (E-mail:Tranquart@med.univ-tours.fr)

Autophagy and Nuclear Changes in FM3A Breast Tumor Cells after Epirubicin, Medroxyprogesterone and Tamoxifen Treatment in vitro

Objective: Autophagy is a form of physiological programmed cell death which is observable after hormonal withdrawal. In this study, the FM3A murine breast tumor cell line was treated with epirubicin alone and with medroxyprogesterone acetate (MPA) or tamoxifen, to determine if structural and kinetic signs of autophagy may also occur in an enhanced manner during epirubicin sensitization via hormonal agents. Methods: One-week soft agar colony growth, 96-hour values of plating and pulse thymidine labeling and electron microscopical examinations were performed following treatment with MPA and tamoxifen alone or with epirubicin. Results: Tamoxifen induced signs of autophagy, which was enhanced when it was combined with MPA. Epirubicin also induced autophagy of secretory granules, which coalesced to form an intracytoplasmic lumen. Combining MPA with epirubicin enhanced the formation of apoptotic blebs and chromatin fragmentation. When epirubicin was combined with tamoxifen, peculiar nuclear structures were formed. Conclusions: Hormonal agents may modulate anthracycline activity towards specific patterns in eliciting cell death, via autophagy and/or as yet unknown nucleolus-specific interactions.

Sequence effect of epirubicin and paclitaxel treatment on pharmacokinetics and toxicity.

Sequence-dependent clinical and pharmacokinetic interactions between paclitaxel and doxorubicin have been reported. Some data have shown an influence of paclitaxel on epirubicin metabolism, but no data are available about the effect of diverse sequences of these drugs. We investigated whether reversing the sequence of epirubicin and paclitaxel affects the pattern or degree of toxicity and pharmacokinetics. Patients receiving epirubicin 90 mg/m(2) by intravenous bolus followed by paclitaxel 175 mg/m(2) over 3-hour infusion or the opposite sequence every 3 weeks for four cycles were eligible. Toxicity was recorded at nadir. Pharmacokinetic data were evaluated at the first and the second cycle and were correlated with toxicity parameters. Thirty-nine consecutive stage II breast cancer patients were treated. Twenty-one patients received epirubicin followed by paclitaxel (ET group), and 18 received the opposite sequence (TE group). No significant difference in nonhematologic toxicity was seen. A lower neutrophil and platelet nadir and a statistically significant slower neutrophil recovery was observed in the TE group. Area under the concentration-time curve (AUC) of epirubicin was higher in the TE group (2,346 ng/mL. h v 1,717 ng/mL. h; P =.002). An inverse linear correlation between epirubicin AUC and neutrophil recovery was also observed (P =.012). No difference was detected in paclitaxel pharmacokinetics. Our results support a sequence-dependent effect of paclitaxel over epirubicin pharmacokinetics that is associated with increased myelotoxicity. Because schedule modifications of anthracyclines and paclitaxel can have clinical consequences, the classical way of administration (ie, anthracyclines followed by paclitaxel) should be maintained in clinical practice.