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ObjectiveTo evaluate the safety, efficacy and therapeutic mechanism of BI 655064, an antagonistic anti-CD40 monoclonal antibody, in patients with rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX-IR).MethodsIn total, 67 patients were randomised to receive weekly subcutaneous doses of 120 mg BI 655064 (n=44) or placebo (n=23) for 12 weeks. The primary endpoint was the proportion of patients who achieved 20% improvement in American College of Rheumatology criteria (ACR20) at week 12. Safety was assessed in patients who received at least one dose of study drug.ResultsAt week 12, the primary endpoint was not met, with 68.2% of patients treated with BI 655064 achieving an ACR20 vs 45.5% with placebo (p=0.064); using Bayesian analysis, the posterior probability of seeing a difference greater than 35% was 42.9%. BI 655064 was associated with greater changes in CD40–CD40L pathway-related markers, including reductions in inflammatory and bone resorption markers (interleukin-6, matrix metalloproteinase-3, receptor activator of nuclear factor-κB ligand), concentration of autoantibodies (immunoglobulin [Ig]G rheumatoid factor [RF], IgM RF, IgA RF) and CD95+ activated B-cell subsets. No serious adverse events (AEs) related to BI 655064 treatment or thromboembolic events occurred; reported AEs were mainly of mild intensity.ConclusionAlthough blockade of the CD40–CD40L pathway with BI 655064 in MTX-IR patients with RA resulted in marked changes in clinical and biological parameters, including reductions in activated B-cells, autoantibody production and inflammatory and bone resorption markers, with a favourable safety profile, clinical efficacy was not demonstrated in this small phase IIa study.Trial registration numberNCT01751776

Background: Neutrophil elastase (NE) is increased in COPD sputum and bronchoalveolar lavage fluid and correlates with clinical symptoms. NE inhibition (NEi) may reduce tissue damage and inflammation. Methods: We conducted two Phase I trials: NCT03588390 (single-blind, partially randomised, placebo-controlled, parallel-group) and NCT03802331 (open-label, randomised, single-dose, two-way crossover); in subjects aged 18–45 receiving BI 1323495 orally, as single rising doses of 10‒600mg (NCT03588390) or two single 100mg doses (NCT03802331). Primary endpoints were number of subjects with investigator-defined drug-related adverse events (DRAEs) (NCT03588390), AUC0–tz and Cmax (NCT03802331). Results: 63 subjects entered NCT03588390; 12 entered NCT03802331. DRAEs were reported in 12 (NCT03588390) and 1 (NCT03802331) subjects; all were mild or moderate and resolved by trial end. Headache was the most common DRAE in subjects receiving BI 1323495 (6 subjects in NCT03588390 and 1 in NCT03802331). NCT03588390 established a pharmacokinetic/pharmacodynamic relationship. A drug concentration of ~70nmol/L achieved 95% NEi in zymosan-stimulated blood. A linear relationship between dose and exposure was demonstrated by a power model in doses ≤300mg (Figure); exposure was higher when administered with food. Conclusions: BI 1323495 was well tolerated in doses up to 600mg, with dose linearity up to 300mg. Exposure was higher in fed than fasted state.

BI 655064 is a humanized antagonistic anti‐cluster of differentiation (CD) 40 monoclonal antibody that selectively blocks the CD40‐CD40L interaction. The CD40‐CD40L pathway is a promising treatment target for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis. The safety, tolerability, pharmacokinetics, and pharmacodynamics of repeated once‐weekly BI 655064 subcutaneous dosing over 4 weeks were evaluated in a multiple‐dose study in healthy subjects. Subjects (N = 40) were randomized 4:1 to four sequential BI 655064 dose groups (80, 120, 180, 240 mg) or to placebo. Safety and tolerability, plasma exposure, CD40 receptor occupancy, and CD40L‐induced CD54 upregulation were assessed over 64 and 78 days for the 80‐ to 180‐mg and 240‐mg dose groups, respectively. BI 655064 exposure increased in a supraproportional manner, due to target‐mediated drug clearance, for doses between 80 mg and 120 mg, but was near proportional for doses greater than 120 mg. Terminal half‐life ranged between 6 and 8 days. Dose‐dependent accumulation of BI 655064 supports the use of a loading dose in future clinical studies. Following 4 weeks of dosing, >90% CD40 receptor occupancy and inhibition of CD54 upregulation were observed at all dose levels, lasting for 17 days after the last dose. BI 655064 was generally well tolerated. There were no serious adverse events and the frequency and intensity of adverse events were similar for BI 655064 and placebo; no dose relationship or relevant signs of an acute immune reaction were observed. These findings support further investigation of BI 655064 as a potential treatment for autoimmune diseases.

Background — Aim: HER2 and TOP2A gene status are of prognostic/predictive relevance and are broadly determined with fluorescent in situ hybridization (FISH). For this purpose, probes against (A) 17p11.1-q11.1 (>5Mb, CENtromere), (B) 17q12 (600Kb, including HER2 among other genes), and (C) 17q21-22 (500Kb, similarly including TOP2A) are used, and gene pathology is determined by B/A and C/A signal ratios according to consensus cut-offs. However, chromosome 17 (chr17) and 17q pathology is complex and may be misinterpreted by this approach, especially in cases with low copy gains. Herein, we profiled A, B and C signals in 1027 adjuvantly treated, early breast cancer patients. Methods: Maximal A, B, C copy numbers (raw FISH data) were submitted to hierarchical clustering as continuous variables. Clusters were compared with clinicopathological parameters, conventional FISH ratios, and were evaluated for their prognostic significance (overall survival, OS) with respect to tumor Ki67 status (13% cut-off), TopoIIa protein (5% cut-off) and ER/PgR positivity (1% cut-off). Results: Clustered values resulted into 832 (81%) putatively chr17 stable tumors (ABClow) and 195 17q unstable tumors. Out of these, 43 (4%) had possible chr17 gain (ABChigh), 30 (3%) HER2/TOP2A gain (BChigh), and 122 (12%) HER2 gain (Bhigh). Unstable 17q was more frequent in Ki67 high, ER/PgR negative, high-grade tumors, and were almost exclusively present in luminal-HER2 and HER2-enriched tumors (all Pearson's p's Conclusions: Profiles of 17p11-q11, 17q12 and 17q21-22 copies according to broadly used FISH probes reveal distinct subgroups of breast cancer with possibly different versions of chromosomal instability that are only partially concordant with classical CEN17, HER2 and TOP2A gain status. The prognostic significance of these profiles seems to depend on tumor proliferation and hormonal status, as well as on the extent of the affected areas on 17q. Validation of these profiles for prospective application may aid in a more accurate assessment of early high-risk breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-15.

The aim of the study was to evaluate the association of vascular endothelial growth factor (VEGF) genotypes with treatment efficacy in a randomized trial. This study compared two chemotherapy regimens (FOLFIRI versus XELIRI) combined with bevacizumab, as first-line treatment for metastatic colorectal cancer. DNA was extracted from blood samples of 173 patients participating in the trial. Genotyping was performed for selected SNPs (VEGF-1154, +936, -634, -2578 and -1498). All candidate genotypes were evaluated for associations with overall survival (OS), progression-free survival (PFS) and response rate (RR). There were no significant differences with respect to the distribution of genotypes in the treatment groups. The VEGF-1154 GG genotype was more frequent in patients not responding to treatment compared with responders (65.5 versus 39.8%, P=0.032). Furthermore, the VEGF-1154 GG genotype was associated with inferior median OS compared with GA (hazards ratio=1.68; 95% confidence interval: 1.10-2.57; P=0.016) or with the alternative genotypes (GA and AA) combined (hazards ratio=1.62; 95% confidence interval: 1.09-2.40; P=0.017). In multivariate analysis, the VEGF-1154 GG genotype remained a significant adverse factor for OS. Our results support the potential predictive ability of VEGF genotypes in patients with metastatic colorectal cancer receiving irinotecan-based chemotherapy plus bevacizumab, in terms of RR and OS. However, current results should be validated prospectively, in larger cohorts.The Pharmacogenomics Journal advance online publication, 16 August 2011; doi:10.1038/tpj.2011.37. Pharmacogenomics J

International audience; Trastuzumab (T) is effective in metastatic breast cancer (MBC) with HER2 overexpression and/or amplification, but resistance to T develops in a significant number of HER2-positive patients. Understanding the mechanisms of resistance is critical to the care of these patients. Formalin-fixed paraffin-embedded tumor tissue samples were collected from 256 patients with T-treated MBC. Clinical information was collected retrospectively from the patients' medical records. Central review of HER2 status by fluorescent in situ hybridization (FISH) and/or immunohistochemistry (IHC) revealed that of the 227 eligible patients only 139 (61%) were truly HER2-positive. PTEN, ER, PgR, and Ki67 were evaluated by IHC, while PTEN status was evaluated by FISH as well. PIK3CA mutations were identified with single nucleotide polymorphism (SNP) genotyping. Median time to progression (TTP) was 14.4 months for the HER2-positive and 10.3 for the HER2-negative patients (log-rank, = 0.22). Survival from the initiation of T (survivalT) was 50.4 months for the HER2-positive and 35.3 for the HER2-negative subgroups ( = 0.006). Higher risk of progression was associated with HER2-positive status and the presence of PIK3CA mutations ( = 0.014). PTEN loss, as determined by IHC, was associated with lower survivalT in the whole population ( = 0.029) and in the HER2-positive population ( = 0.017). PIK3CA mutations and/or PTEN loss status were evaluated together as a single parameter, to estimate the impact of activation of the PI3K/AKT molecular pathway, and it was significantly associated with both decreased TTP ( = 0.003 in the total population, = 0.004 in HER2-positive patients) and survival (survivalT, = 0.011 in total, = 0.006 in HER2-positive). In this trastuzumab-treated breast cancer population, PIK3CA activating mutations were associated with shorter TTP and PTEN loss with decreased survival. The activation of the PI3K/AKT pathway from either defect was associated with both TTP and survival, indicating the adverse effect of this pathway's status on trastuzumab efficacy.

Several studies have recently indicated the prognostic or predictive role of several biomarkers in colorectal cancer. We sought to investigate the prognostic value of prostaglandin synthase 2 (PTGS2), cyclooxygenase 2 (COX2), thymidylate synthetase (TYMS), thymidine phosphorylase (TYMP), dihydropyrimidine dehydrogenase (DPYD) and topoisomerase I (TOPO1) in colorectal cancer patients treated with 5-FU-based regimens, such as De Gramont and FOLFOX in the adjuvant setting.In total, 96 formalin-fixed paraffin-embedded and 30 fresh-frozen tumor tissue samples were evaluated using immunohistochemistry, quantitative reverse transcription-polymerase chain reaction and microarray gene expression profiling, respectively.The majority of tumors exhibited protein overexpression of COX2 (69%), TYMS (75%) and TOPO1 (75%). There was a significant association of TYMP protein expression with T classification, gender and stage (p=0.040, p=0.041 and p=0.011, respectively). TOPO1 protein expression was correlated with TOPO1 mRNA expression and was positively associated with stage (p=0.002) and lymph node infiltration (p=0.004). In univariate analysis, patients with high TYMS mRNA expression were shown to have a significantly lower risk for progression and death (Wald's p=0.030 and p=0.015, respectively). However, in multivariate analysis, only a trend for decreased risk for death was shown in patients with high TYMS mRNA expression (Wald's p=0.083), while patients with high PTGS2 mRNA expression had a trend for lower risk for progression (p=0.064). Using supervised hierarchical clustering, based on the expression in fresh-frozen tumor tissue of PTGS2, TYMS, TYMP and DPYD, our 30 patients were separated into two clusters. One of the clusters was enriched with patients with infiltrated lymph nodes (p0.05), suggesting that these genes might have an impact on the tumor's ability to metastasize.These findings indicate a possible prognostic role of TYMS mRNA expression and highlight a cluster of genes associated with nodal metastases that warrant further investigation in a larger cohort of patients with colorectal cancer treated with 5-FU-based adjuvant chemotherapy.

To investigate the immunohistochemical expression of p85 in a cohort of trastuzumab-treated HER2-positive and HER2-negative metastatic breast cancer patients. The medical records of all patients with metastatic breast cancer treated with trastuzumab-based regimens between 1998 and 2010 were reviewed and clinical information was obtained. Formalin-fixed paraffin-embedded tumor tissue samples with adequate material were retrospectively collected from 183 patients. Samples were evaluated by immunohistochemistry for p85, estrogen receptors (ER), progesterone receptors (PgR), HER2, Ki67, PTEN and phosphorylated Akt (S473 and T308). HER2 status was studied by fluorescence in situ hybridization, as well. PIK3CA mutational status was also evaluated. Median follow-up for all patients was 72 months. Central re-evaluation for HER2 revealed only 111 HER2-positive cases, with the remaining 72 patients being HER2-negative. Median survival was longer in HER2-positive patients (50.7 months) compared to HER2-negative patients (36.6 months) both treated with trastuzumab, but this difference has not reached significance (p = 0.068). In total, 62% of the patients were found positive for p85, however the p85 protein was not found to be differentially expressed in HER2-positive versus HER2-negative cases. There were no significant associations between protein expression of p85 and any of the markers under study, or with time to progression. Positive p85 protein expression was however associated with poor survival in trastuzumab-treated HER2-positive patients. In our cohort of trastuzumab-treated HER2-positive breast cancer patients, positive p85 protein expression appears to be a prognostic factor of poor survival and, if validated, might have important implications in the treatment of such patients.

There are scarce data available on the prognostic/predictive value of p-Akt and p-mTOR protein expression in patients with high-risk early breast cancer. Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 997 patients participating in two adjuvant phase III trials were assessed for EGFR, PTEN, p-Akt, p-mTOR protein expression, and PIK3CA mutational status. These markers were evaluated for associations with each other and with selected patient and tumor characteristics, immunohistochemical subtypes, disease-free survival (DFS), and overall survival (OS). p-mTOR protein expression was negatively associated with EGFR and positively associated with PTEN, with p-Akt473, and with the presence of PIK3CA mutations. EGFR expression was positively associated with p-Akt473, p-Akt308, and PIK3CA wild-type tumors. Finally, p-Akt308 was positively associated with p-Akt473 expression. In univariate analysis, EGFR (p = 0.016) and the coexpression of EGFR and p-mTOR (p = 0.015) were associated with poor OS. Among patients with p-Akt308-negative or low-expressing tumors, those treated with hormonal therapy were associated with decreased risk for both relapse and death (p = 0.013 and p

Background The oxaliplatin (ΟΧΑ)-based regimens FOLFOX and XELOX can cause peripheral neuropathy. It is unknown if ΟΧΑ, alone or in combination regimens, affects the Autonomous Nervous System (ANS). Accordingly, we evaluated the impact of ΟΧΑ-based chemotherapy on the ANS. Methods We enrolled 36 patients with colorectal cancer, treated with adjuvant mFOLFOX6 or XELOX chemotherapy, and 22 healthy volunteers. For the assessment of ANS function, participants completed a questionnaire and underwent neurophysiological examination at three time points (baseline, 3–4 months and 6–8 months after the first chemotherapy cycle). ANS testing included assessment of the adrenergic cardiovascular function (orthostatic hypotension-OH), parasympathetic heart innervation (ratio 30/15) and Sympathetic Skin Response (SSR). Results The values of the 30/15 ratio were significantly reduced at the two time point assessments compared to baseline (Wilcoxon signed ranks test, both P

The Androgen Receptor (AR) is a potential prognostic marker and therapeutic target in breast cancer. We evaluated AR protein expression in high-risk breast cancer treated in the adjuvant setting. Tumors were subtyped into luminal (ER+/PgR±/AR±), molecular apocrine (MAC, [ER-/PgR-/AR+]) and hormone receptor negative carcinomas (HR-negative, [ER-/PgR-/AR-]). Subtyping was evaluated with respect to prognosis and to taxane therapy. High histologic grade (p 0.001) and increased proliferation (p = 0.001) more often appeared in MAC and HR-negative than in luminal tumors. Patients with MAC had outcome comparable to the luminal group, while patients with HR-negative disease had increased risk for relapse and death. MAC outcome was favorable upon taxane-containing treatment; this remained significant upon multivariate analysis for overall survival (HR 0.31, 95%CI 0.13-0.74, interaction p = 0.035) and as a trend for time to relapse (p = 0.15). In conclusion, AR-related subtyping of breast cancer may be prognostic and serve for selecting optimal treatment combinations.

Tissue microarrays (TMAs) are an attractive alternative to analysis of whole sections (WS). For breast carcinomas, the recent recommendations for cut-offs (i.e. Ki67, H-score) have necessitated the re-evaluation of TMAs.TMA results of immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) testing for Estrogen receptors (ER), Progesterone receptors (PgR), Ki67 and HER2 were compared against the results of WS for 88 breast carcinomas.We found excellent agreement between the two methods for ER and PgR IHC evaluation, using the H-score (Kappa coefficient 0.972 and 0.9, respectively). There was also excellent correlation for HER2 IHC (Kappa coefficient 1) and amplification (Kappa coefficient 0.933). Furthermore, scoring of Ki67 was highly-correlated between TMAs and WS (Kappa coefficient 0.954). The latter excellent correlation has not, to our knowledge, been previously reported.For breast cancer, TMAs are an efficient and reliable alternative to the use of WS, using the currently recommended markers, evaluation protocols and cut-off values.

PURPOSE: Local recurrence is the major manifestation of treatment failure in patients with operable laryngeal carcinoma. Established clinicopathological factors cannot sufficiently predict patients that are likely to recur after treatment. Additional tools are therefore required to accurately identify patients at high risk for recurrence. This study attempts to identify and independently validate gene expression models, prognostic of disease-free survival (DFS) in operable laryngeal cancer. MATERIALS AND METHODS: Using Affymetrix U133A Genechips, we profiled fresh-frozen tumor tissues from 66 patients with laryngeal cancer treated locally with surgery. We applied Cox regression proportional hazards modeling to identify multigene predictors of recurrence. Gene models were then validated in two independent cohorts of 54 and 187 patients (fresh-frozen and formalin-fixed tissue validation sets, respectively). RESULTS: We focused on genes univariately associated with DFS (p

Background The combinations of methotrexate, vinblastine, Adriamycin, cisplatin (Pharmanell, Athens, Greece) (MVAC) or gemcitabine, cisplatin (GC) represent the standard treatment of advanced urothelial cancer (UC). Dose-dense (DD)-MVAC has achieved longer progression-free survival (PFS) than the conventional MVAC. However, the role of GC intensification has not been studied. We conducted a randomized, phase III study comparing a DD-GC regimen with DD-MVAC in advanced UC. Patients and methods One hundred and thirty patients were randomly assigned between DD-MVAC: 66 (M 30 mg/m2, V 3 mg/m2, A 30 mg/m2, C 70 mg/m2 q 2 weeks) and DD-GC 64 (G 2500 mg/m2, C 70 mg/m2 q 2 weeks). The median follow-up was 52.1 months (89 events). Results The median overall survival (OS) and PFS were 19 and 8.5 months for DD-MVAC and 18 and 7.8 months for DD-GC (P = 0.98 and 0.36, respectively). Neutropenic infections were less frequent for DD-GC than for DD-MVAC (0% versus 8%). More patients on DD-GC received at least six cycles of treatment (85% versus 63%, P = 0.011) and the discontinuation rate was lower for DD-GC (3% versus 13%). Conclusions Although DD-GC was not superior to DD-MVAC, it was better tolerated. DD-GC could be considered as a reasonable therapeutic option for further study in this patient population. Clinical Trial Number ACTRN12610000845033, www.anzctr.org.au .

Background BRCA1 (B), ERCC1 (E), RRM1 (R) and TYMS (T) mRNA expression has been extensively studied with respect to NSCLC patient outcome upon various chemotherapy agents. However, these markers have not been introduced into clinical practice yet. One of the reasons seems to be lack of a standard approach for the classification of the reported high/low mRNA expression. The aim of this study was to determine the prognostic/predictive impact of B, E, R, T in routinely-treated NSCLC patients by taking into account the expression of these genes in the normal lung parenchyma. Methods B, E, R, T mRNA expression was examined in 276 NSCLC samples (real-time PCR). The normal range of B, E, R, T transcript levels was first determined in matched tumor – normal pairs and then applied to the entire tumor series. Four main chemotherapy categories were examined: taxanes-without-platinum (Tax); platinum-without-taxanes (Plat); taxanes/platinum doublets (Tax/Plat); and, all-other combinations. Results In comparison to remotely located normal lung parenchyma, B, E, R, T mRNA expression was generally increased in matched tumors, as well as in the entire tumor series. Therefore, tumors were classified as expressing normal or aberrant B, E, R, T mRNA. In general, no marker was associated with overall and progression free survival (OS, PFS). Upon multivariate analysis, aberrant intratumoral TYMS predicted for shorter PFS than normal TYMS in 1st line chemo-naïve treated patients (p = 0.012). In the same setting, specific interactions were observed for aberrant TYMS with Plat and Tax/Plat (p = 0.003 and p = 0.006, respectively). Corresponding patients had longer PFS in comparison to those treated with Tax (Plat: HR = 0.234, 95% CI:0.108-0.506, Wald’s p Conclusion Classifying intratumoral B, E, R, T mRNA expression in comparison to normal lung may facilitate standardization of these parameters for prospective studies. With this approach, NSCLC patients with aberrant intratumoral TYMS expression will probably fare better with platinum-based treatments.

Background The aim was to compare two standard chemotherapy regimens combined with bevacizumab as first-line treatment in patients with metastatic colorectal cancer. Methods Patients previously untreated for metastatic disease were randomized in: group A (irinotecan, capecitabine, bevacizumab, every 3 weeks; XELIRI-bevacizumab) and group B (irinotecan, leucovorin, fluorouracil, bevacizumab, every 2 weeks; FOLFIRI-bevacizumab). Primary endpoint was progression-free survival (PFS). Plasma concentrations of nitric oxide, osteopontin, TGF-β1 and VEGF-A were measured at baseline and during treatment. Results Among 285 eligible patients, 143 were randomized to group A and 142 to group B. Fifty-five patients (38.5%) in group A and 57 (40.1%) in group B responded (p = 0.81). After a median follow-up of 42 months, median PFS was 10.2 and 10.8 months (p = 0.74), while median OS was 20.0 and 25.3 months (p = 0.099), for groups A and B, respectively. Most frequent grade 3–4 toxicities (group A vs group B) were neutropenia (13% vs 22%, p = 0.053) and diarrhea (19% vs 11%, p = 0.082). Baseline plasma osteopontin concentrations demonstrated prognostic significance for both PFS and OS. Conclusions This trial did not show significant differences in efficacy between the groups. However, the toxicity profile was different. Baseline plasma osteopontin concentrations demonstrated independent prognostic significance. (Registration number: ACTRN12610000270011)

Background The vast majority of patients with HER2-positive metastatic breast cancer (MBC) treated with trastuzumab eventually develop resistance to this agent. There is an unmet need therefore, for identifying biological markers with possible prognostic/predictive value in such patients. The aim of this study was to investigate the prognostic role of topoisomerase II alpha gene (TOP2A) amplification and protein (TopoIIa) expression in patients treated with trastuzumab-containing regimens. Methods Formalin-fixed paraffin-embedded tumor tissue samples were retrospectively collected from 225 eligible patients treated with trastuzumab. Protein expression of ER, PgR, Ki67, PTEN, HER2 and TopoIIa were centrally assessed by immunohistochemistry. HER2 and TOP2A gene amplification was evaluated by fluorescence in situ hybridization. PIK3CA mutations were identified by single nucleotide polymorphism genotyping. Survival was evaluated from the initiation of trastuzumab as 1st line treatment to the date of last follow-up or death. Results Among the 225 samples analyzed, only 137 (61%) were found to be HER2-positive. TOP2A was amplified in 41% and deleted in 16% of such tumors. TOP2A gene amplification was more frequent in ER-negative tumors. TopoIIa protein expression was observed in the majority (65%) of the samples and was associated with ER-positive status, high Ki67 expression, presence of PTEN protein and PIK3CA mutations. Median follow-up for patients treated in the 1st line was 51 months. Survival was more prolonged with trastuzumab-containing treatment in HER2-positive patients (50 months, log-rank, p=0.007). TOP2A non-amplified or deleted tumors were associated with increased risk for death compared to TOP2A amplified tumors (HR=2.16, Wald’s p=0.010 and HR=2.67, p=0.009, respectively). In multivariate analysis, a significant interaction of TOP2A with anthracycline treatment (either in the adjuvant or the 1st line setting) was observed for survival (Wald’s p=0.015). Among the TOP2A amplified subgroup, anthracycline-treated patients were associated with decreased risk for death. Conclusions TOP2A gene amplification was shown to be a favorable prognostic marker in HER2-positive MBC patients treated with trastuzumab, such an effect however, appears to rather be related to treatment with anthracyclines (predictive marker for benefit from anthracyclines). The results of the present retrospective study warrant validation in larger cohorts of patients treated in the context of randomized trials.

Background HER2 and TOP2A parameters (gene status, mRNA and protein expression) have individually been associated with the outcome of patients treated with anthracyclines. The aim of this study was to comprehensively evaluate the prognostic/predictive significance of the above parameters in early, high-risk breast cancer patients treated with epirubicin-based, dose-dense sequential adjuvant chemotherapy. Methods In a series of 352 breast carcinoma tissues from patients that had been post-operatively treated with epirubicin-CMF with or without paclitaxel, we assessed HER2 and TOP2A gene status (chromogenic in situ hybridization), mRNA expression (quantitative reverse transcription PCR), as well as HER2 and TopoIIa protein expression (immunohistochemistry). Results HER2 and TOP2A amplification did not share the same effects on their downstream molecules, with consistent patterns observed in HER2 mRNA and protein expression according to HER2 amplification (all parameters strongly inter-related, p values < 0.001), but inconsistent patterns in the case of TOP2A. TOP2A gene amplification (7% of all cases) was not related to TOP2A mRNA and TopoIIa protein expression, while TOP2A mRNA and TopoIIa protein were strongly related to each other (p < 0.001). Hence, TOP2A amplified tumors did not correspond to tumors with high TOP2A mRNA or TopoIIa protein expression, while the latter were characterized by high Ki67 scores (p = 0.003 and p < 0.001, respectively). Multivariate analysis adjusted for nodal involvement, hormone receptor status, Ki67 score and HER2/TOP2A parameters revealed HER2/TOP2A co-amplification (21.2% of HER2 amplified tumors) as an independent favorable prognostic factor for DFS (HR = 0.13, 95% CI: 0.02-0.96, p = 0.046); in contrast, increased HER2/TOP2A mRNA co-expression was identified as an independent adverse prognostic factor for both DFS (HR = 2.41, 95% CI: 1.31-4.42, p = 0.005) and OS (HR = 2.83, 95% CI: 1.42-5.63, p = 0.003), while high TOP2A mRNA expression was an independent adverse prognostic factor for OS (HR = 2.06, 95% CI: 1.23-3.46, p = 0.006). None of the parameters tested was associated with response to paclitaxel. Conclusions This study confirms the favorable prognostic value of HER2/TOP2A co-amplification and the adverse prognostic value of high TOP2A mRNA expression extending it to the adjuvant treatment setting in early high-risk breast cancer. The strong adverse prognostic impact of high HER2/TOP2A mRNA co-expression needs further validation in studies designed to evaluate markers predictive for anthracyclines. Trial registration Australian New Zealand Clinical Trials Registry ACTRN12611000506998.

To explore the impact of dose intensity (DI) in the adjuvant setting of breast cancer, a randomized phase III trial was conducted comparing postoperative dose-dense sequential chemotherapy with epirubicin, paclitaxel, and cyclophosphamide, methotrexate and fluorouracil (CMF)in high-risk breast cancer patients. From Oct 2000 to June 2005, 1,121 node-positive patients were randomized to dose-dense sequential epirubicin 110 mg/m(2) and paclitaxel (Taxol((R)), Bristol Myers-Squibb, Princeton, NJ) 250 mg/m(2) (group A), or concurrent epirubicin 83 mg/m(2) and paclitaxel 187 mg/m(2) (group B), both followed by three cycles of "intensified" combination chemotherapy with CMF. By protocol design total cumulative dose and duration of treatment were identical in both groups. Dose intensity of epirubicin and paclitaxel was double in the dose-dense arm. Prophylactic treatment with granulocyte colony-stimulating factor was given with the dose-dense treatments. Disease-free survival (DFS) was the primary endpoint. At a median follow-up of 76 months, 253 patients (23%) had documented disease relapse (123 vs. 130 in groups A and B, respectively) and 208 deaths (101, group A and 107, group B) had been observed. The 5-year DFS rate of 74 and 74% and OS rate of 86 and 85% were observed for group A and group B, respectively. No differences were found in DFS or OS between the two treatment groups (P = 0.78 and P = 0.45 for DFS and OS, respectively). Safety analysis results showing that both regimens were well tolerated and safe have been previously published (Fountzilas et al. Ann Oncol 2008). No DFS or OS benefit from the dose-dense sequential epirubicin and paclitaxel was detected when compared to the concurrent administration of the same drugs. No additional safety issues were raised with long-term follow-up. Breast Cancer Res Treat

BACKGROUND: Paclitaxel (Taxol) and vinorelbine have shown synergism of cytotoxic effects in vitro and clinical activity in phase I and II studies. This combination was compared prospectively with the paclitaxel/gemcitabine regimen in non-operable non-small-cell lung cancer. PATIENTS AND METHODS: Chemotherapy-naive patients, stage IIIbwet and IV with performance status (0-1), were randomized to receive paclitaxel 200 mg/m(2) on day 1 plus gemcitabine 1 gm/m(2) (group A) on days 1 and 8 every 3 weeks or paclitaxel 80 mg/m(2) plus vinorelbine 22.5 mg/m(2) (group B) on days 1, 8 and 15 every 4 weeks. RESULTS: A total of 398 out of 415 patients were eligible for analysis on intent-to-treat basis (group A: 196, group B: 202). Progression-free survival (PFS) was 5.0 months [95% confidence interval (CI) 4.3-5.6] and 4.4 months (95% CI 3.7-5.2) for groups A and B respectively (P=0.365). Median survival was 11.1 months (95% CI 9.2-13.0) and 8.6 months (95% CI 7.0-10.2) for groups A and B respectively (P = 0.147). Grade 3/4 neutropenia and leukopenia were worse in group B (P

Background: To analyse the discriminative impact of osteopontin (OPN) and activated leukocyte cell adhesion molecule (ALCAM), combined with human epidermal growth factor 2 (HER2) and oestrogen receptor (ER) in breast cancer. Methods: Osteopontin, ALCAM, HER2 and ER mRNA expression in breast cancer tissues of 481 patients were analysed (mRNA microarray analysis, kinetic RT–PCR). Hierarchical clustering was performed in training cohort A (N=100, adjuvant treatment) and validation cohorts B (N=200, no adjuvant treatment, low-risk) and C (N=181, adjuvant treatment, high-risk). Results: Negative/low ER and HER2, high OPN and low ALCAM mRNA expression helped to identify patients at particularly high risk, showing shorter DFS, P

In the metastatic setting, a detected time trend to improved prognosis could be attributed to the corresponding recent advances in the therapeutic approaches. The aim of the current study was to first assess, in a large cohort of well over a thousand patients, the time trends in survival in MBC for the last 15 years and second to explore its association to prognostic factors affecting outcome including therapeutic regimen. This meta-analysis uses individual patient data collected from all the trials on MBC (6 nonrandomized, 4 randomized) conducted by HeCOG from 1991 through 2006. Four 4-year time periods (1991–1994, 1995–1998, 1999–2002, and 2003–2006) were constructed for exploration of time trends in survival according to the patient’s date of metastatic diagnosis. Different first line regimens in the 10 trials include anthracycline monotherapy (epirubicin, in the early 1990s) and taxane-containing regimens either as monotherapy or in different combinations with anthracyclines or other drugs. In two phase II studies and in the last randomized study, trastuzumab was administered in all the patients with HER2 overexpressing tumors. In this study, information is based on a total of 1361 patients with a median follow up of 3.7 years and median survival of 1.9 years (median survival 1.28, 1.68, 2.20, and 2.57 years for 1991–1994, 1995–1998, 1999–2002, and 2003–2006, respectively). Survival improved significantly across diagnosis time periods, by 25, 44, and 51%, respectively, in each time period (1995–1998: HR = 0.75, P = 0.004; 1999–2002: HR = 0.56, P

Bayesian analysis of correlated binary data when individual information is not available is considered. In particular, a binary outcome is measured on the same subjects of two independent groups at two separate occasions (usually time points). The groups are formulated through a binary exposure or a prognostic factor. Interest lies in estimating the association between exposure and outcome over time. Standard methods for this purpose apply on the individual item responses and are insufficient in case these are missing. Moreover it is assumed that the only available information is the marginal 2x2 cross-tabulations between the grouping variable and the response for each occasion. Assuming independent binomial distributions for the two groups, the success probabilities for each occasion as well as the associations between exposure and outcome, based on the corresponding odds ratios, are estimated. In order to deal with the missing information of each item's response and to estimate the corresponding transition probabilities, a Bayesian procedure is adopted. Computational Statistics & Data Analysis

e19048 Background: KRAS mutations are reported in 20-25% of non-small cell lung cancer (NSCLC). Little is known about the prognostic/predictive role of KRAS in advanced NSCLC, with conflicting results among small studies. Recent evidence showed that KRAS mutations could predict for worse outcome in patients treated with platinum-based adjuvant chemotherapy. Methods: KRAS and EGFR genotypes were evaluated in 414 NSCLC patients with available clinical data, diagnosed from March 2000 to December 2012 (tissue blocks from the HeCOG tumor repository). KRAS and EGFR mutations were associated with clinicopathological parameters (mutated vs. wild-type). Outcome comparisons were performed in 214 metastatic patients with treatment data available, following 1st-line chemotherapy without tyrosine kinase inhibitors. Results: The majority of the patients were male (80%), current smokers (53%), with adenocarcinoma (AC) histology (65%). EGFR mutations were found in 10% and KRAS mutations in 17% of all histological types, while in AC they were 14% and 21%, respectively. Most EGFR mutations were classical (79%), while most common KRAS mutations were p.G12C (39%), p.G12D (30%) and p.G12V (15%). Two tumors had concurrent EGFR and KRAS mutations. EGFR mutations were significantly associated with female gender, AC histology and non-smoking status, as previously described. KRAS mutations were associated with AC histology and younger age (st-line treatment decreases the risk of death.

Background Dose-dense sequential chemotherapy including anthracyclines and taxanes has been well established in the adjuvant setting of high-risk operable breast cancer. However, the preferable taxane and optimal schedule of administration have not been defined, as yet. Patients and methods From July 2005 until November 2008, 1,001 patients (990 eligible) were randomized to receive 3 cycles of epirubicin 110 mg/m2 followed by 3 cycles of paclitaxel 200 mg/m2 followed by 3 cycles of CMF (cyclophosphamide 840 mg/m2; methotrexate 57 mg/m2; fluorouracil 840 mg/m2) with G-CSF support (Group A; 333 patients) or to 3 cycles of epirubicin followed by 3 cycles of CMF, as in Group A, followed 3 weeks later by 9 weekly cycles of docetaxel 35 mg/m2 (Group B; 331 patients) or 9 weekly cycles of paclitaxel 80 mg/m2 (Group C; 328 patients). Radiation and hormonal therapy were given after the completion of chemotherapy. Trastuzumab was administered for 1 year to all HER2-positive patients post radiation. Results At a median follow-up of 60 months, 123 patients had documented disease relapse (51 in Group A, 37 in Group B and 35 in Group C) and 81 deaths (30 in group A, 22 in group B and 29 in group C) had been observed. The 3-year disease-free survival (DFS) rate was 86%, 91% and 89%, with overall survival (OS) rates of 96%, 97% and 96%, respectively. No differences were found in DFS or OS between the three treatment groups (log-rank, p = 0.38 and p = 0.48, respectively). The most frequently reported severe adverse events were neutropenia (29% vs 27% vs 24%, p = 0.31) and leucopenia (12% vs 13% vs 10%, p = 0.66). Febrile neutropenia occurred in fifty patients (6%, vs 5% vs 5%, p = 0.81). Severe mucositis was more frequent in Group B (3% vs 6% vs 1%, p = 0.001), while severe neuropathy was more frequent in Group A (4% vs 0% vs 1%, p Conclusions No significant differences in DFS and OS between the three regimens were identified. Taxane regimen and schedule of administration preferentially influenced the type of severe toxicities. HER2-positive patients demonstrated comparable 3-year DFS and OS rates with those reported in other similar studies. Disclosure All authors have declared no conflicts of interest.

5033 Background: The combination of carboplatin/paclitaxel represents the standard 1st-line chemotherapy in advanced OC, FC, and PPC. The optimal duration of paclitaxel treatment has not been defined, while its use is associated with cumulative neurotoxicity in about 50% of patients, which becomes long-term in 15-20% of cases. We, therefore, designed a randomized study to investigate the effect of administering 4 instead of 8 cycles of paclitaxel in the combination carboplatin/paclitaxel on efficacy and tolerability of this treatment. Methods: Patients with FIGO stages IIC-IV OC, FC, PPC were included. Carboplatin AUC 6 and paclitaxel at 175 mg/m2 were used. Both agents were administered for 8 cycles in the CP8 arm, while paclitaxel was administered only for 4 cycles in the C8P4. The study was powered to detect a ± 15% difference in survival rate to a baseline rate of 50 % at the 3-year time point. Results: 389 pts were randomized (2/2004-1/2008) and 380 were eligible for analysis (CP8: 192, C8P4:188). The distribution (CP8 vs C8P4) of baseline characteristics were: stage III: 78% vs. 76%; IV: 12% vs. 15%, residual disease 0 cm: 25% vs. 22%, ECOG PS 0: 68% vs. 64%, serous carcinomas: 79% vs. 68%, tumor grade III: 56% vs. 63%. During a median follow up of 72.3 months 231 patients (111 [58%] in CP8 arm and 120 [64%] in the C8P4 arm) have died. Median PFS was significantly shorter in the C8P4 arm (21.41 vs. 16.46 months, HR [95% CI]: 1.36 [1.07-1.71], Wald’s p=0.011), while OS was similar between the two arms (53.41 vs. 46.59 months, HR [95% CI]: 1.18 [0.91-1.53], Wald’s p=0.211). Lower grade 3 or 4 neurotoxicity (1.9% vs. 10.8%, p< 0.001) but higher myelotoxicity (neutropenia 38.8% vs. 28.8%, p=0.031; thrombocytopenia 20% vs. 8.3%, p=0.004) was observed in the C8P4 arm. Conclusions: Lowering the total number of cycles of paclitaxel in 1st-line chemotherapy of advanced OC, FC, PPC resulted in similar OS but shorter median PFS and is not recommended in this setting. The reduction of neurotoxicity by limiting the total paclitaxel cycles to 4 is achieved at the expense of higher myelotoxicity.

5505 Background: Local recurrence is the major manifestation of treatment failure in patients with operable laryngeal carcinoma. Established clinicopathological factors cannot sufficiently predict ...

Background In this study several tumor-related volumes were assessed by means of a computer-based application and a survival analysis was conducted to evaluate the prognostic significance of pre- and postoperative volumetric data in patients harboring glioblastomas. In addition, MGMT (O6-methylguanine methyltransferase) related parameters were compared with those of volumetry in order to observe possible relevance of this molecule in tumor development. Methods We prospectively analyzed 65 patients suffering from glioblastoma (GBM) who underwent radiotherapy with concomitant adjuvant temozolomide. For the purpose of volumetry T1 and T2-weighted magnetic resonance (MR) sequences were used, acquired both pre- and postoperatively (pre-radiochemotherapy). The volumes measured on preoperative MR images were necrosis, enhancing tumor and edema (including the tumor) and on postoperative ones, net-enhancing tumor. Age, sex, performance status (PS) and type of operation were also included in the multivariate analysis. MGMT was assessed for promoter methylation with Multiplex Ligation-dependent Probe Amplification (MLPA), for RNA expression with real time PCR, and for protein expression with immunohistochemistry in a total of 44 cases with available histologic material. Results In the multivariate analysis a negative impact was shown for pre-radiochemotherapy net-enhancing tumor on the overall survival (OS) (p = 0.023) and for preoperative necrosis on progression-free survival (PFS) (p = 0.030). Furthermore, the multivariate analysis confirmed the importance of PS in PFS and OS of patients. MGMT promoter methylation was observed in 13/23 (43.5%) evaluable tumors; complete methylation was observed in 3/13 methylated tumors only. High rate of MGMT protein positivity (> 20% positive neoplastic nuclei) was inversely associated with pre-operative tumor necrosis (p = 0.021). Conclusions Our findings implicate that volumetric parameters may have a significant role in the prognosis of GBM patients. Furthermore, volumetry could help not only to improve the prediction of outcome but also the outcome itself by identifying patients at high risk of treatment failure and, thus, seek alternative treatment for these patients. In this small series, MGMT protein was associated with less aggressive tumor characteristics.

Background alphaB-crystallin is a small heat shock protein that has recently been characterized as an oncoprotein correlating with the basal core phenotype and with negative prognostic factors in breast carcinomas. The purpose of this study was to evaluate alphaB-crystallin with respect to clinicopathological parameters and the outcome of patients with operable high-risk breast cancer. Methods A total of 940 tumors were examined, derived from an equal number of patients who had participated in two randomized clinical trials (paclitaxel-containing regimen in 793 cases). Immunohistochemistry for ER, PgR, HER2, Ki67, CK5, CK14, CK17, EGFR, alphaB-crystallin, BRCA1 and p53 was performed. BRCA1 mutation data were available in 89 cases. Results alphaβ-crystallin was expressed in 170 cases (18.1%) and more frequently in triple-negative breast carcinomas (TNBC) (45% vs. 14.5% non-TNBC, p 30% positive neoplastic cells, was associated with adverse overall survival (Wald’s p = 0.046). However, alphaB-crystallin was not an independent prognostic factor upon multivariate analysis. No interaction between taxane-based therapy and aβ-crystallin expression was observed. Conclusions In operable high-risk breast cancer, alphaB-crystallin protein expression is associated with poor prognostic features indicating aggressive tumor behavior, but it does not seem to have an independent impact on patient survival or to interfere with taxane-based therapy. Trial registrations ACTRN12611000506998 (HE10/97 trial) and ACTRN12609001036202 (HE10/00 trial).