We retrospectively investigated in women treated with fulvestrant for HR+/HER2 negative advanced breast cancer clinical, pathological and molecular features associated with long-term benefit from treatment defined as being progression-free at 18 months. Specifically, we analyzed on formalin-fixed paraffin-embedded tumor samples ESR1 and PI3KCA mutations and miRNAs profiles. 59 patients were evaluable (median age of 67 years, range 32–92). 18-month PFS rate was 27%; the lack of visceral metastases significantly predicted the likelihood of being progression-free at 18 months, while PI3KCA mutations, found in 36% of patients, were not associated with 18-month PFS. As of miRNAs, miR-549a, miR-644a, miR-16-5p were negatively while let-7c-5p was positively associated with 18-month PFS. In addition, miR-520d-3p and miR-548g-3p values were significantly lower while miR-603, miR-181a-5p and miR-199a-miR-199b-3p values were significantly higher in patients achieving 18-month PFS. In silico analysis of targets modulated by these two latter groups of miRNAs show that in patients achieving 18-month PFS the Hippo and Wnt signaling pathways were predicted to be upregulated while endocrine resistance was potentially repressed by miR-603, miR-181a-5p and miR-199a-miR-199b-3p. Our results provide additional clues on the molecular mechanisms involved in fulvestrant activity and resistance. Underlying pathways should be further elucidated and confirmed in larger cohorts.

e14611 Background: Retrospective studies showed that efficacy of C increases in molecularly-defined subsets of KRAS wt mCRC pts. In pretreated pts, biweekly C seems as effective as a weekly schedule. Aim of this field-practice study was to evaluate the efficacy of FOLFIRI plus biweekly C (C-FOLFIRI) as first-line treatment for mCRC and prospectively validate PTEN expression as a prognostic marker. Methods: Pts with previously untreated metastatic KRAS wt mCRC not suitable for curative-intent resection were eligible. Initially, pts were randomized to receive FOLFIRI alone or in combination with C (500 mg/m2 every two weeks). After a protocol amendment, pts were assigned to receive only C-FOLFIRI, while the FOLFIRI alone arm was discontinued. Clinical outcome was assessed according to expression of PTEN and MET by immunohistochemistry (IHC), as well as to BRAF mutations. Results: From April, 2009 to June, 2012, a total of 89 pts were enrolled. 35 pts were randomly assigned to receive FOLFIRI alone, while 54 pts received C-FOLFIRI: 33 were treated in the random phase and 21 after protocol amendment. Median overall survival (OS) was 17.7 months for FOLFIRI and 23.3 months for C-FOLFIRI. Corresponding median progression-free survival was 8.2 and 6.8 months, respectively. Objective response rate was 52.0% for FOLFIRI and 52.3% for C-FOLFIRI. The toxicity profile of C-FOLFIRI was consistent with that expected from a weekly C schedule. 23/38 pts (61.0%) assessed for PTEN were positive for IHC expression, 8/39 pts (21.0%) had a MET high status, 6/45 pts (13.3%) had BRAF mutations. In pts treated with C-FOLFIRI, only BRAF mutations negatively affected OS (Hazard ratio mutations vs. wt: 4.77; 95%CI 1.41-16.13; p = 0.01). Conclusions: Safety, effectiveness and generalizability of C-FOLFIRI validate in field practice results gained with FOLFIRI plus a weekly C schedule. Despite the small sample size, analysis of BRAF mutations reaffirms the strong prognostic impact of this biomarker. The extended analysis of BRAF mutations, PTEN and MET expression on all pts is ongoing. Data will be presented. Clinical trial information: NCT01068132.

7585 Background: Thymidylate synthase (TS) expression has been reported to predict pem activity in pts with advanced NSCLC. Besides TS, pem inhibits multiple enzymes of the folate pathway including dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GART) and aminoimidazole carboxamide ribonucleotide formyltransferase (AICAR). Aim of present study was to investigate whether these or other biomarkers influence pem activity in NSCLC pts. Methods: Advanced pretreated NSCLC pts who received at least two cycles of single agent pem were considered eligible if they had available tumor tissue to assess at least one of the proposed biomarkers. TS, DHFR, GART and AICAR protein expression was assessed by immunohistochemistry (IHC) in FFPE tumor samples. Pts were grouped as IHC+ and IHC– according to staining intensity. Additionally, presence of EGFR and KRAS mutations was investigated. Results: Ninety-six pts were included in the study. The majority of subjects was male (72%), smokers (93%), with adenocarcinoma (72%). Response rate (RR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS) were 12%, 47%, 2.3 and 9.5 months, respectively. Most pts resulted TS-(72%), AICAR-(82%), DHFR+ (68%), GART- (61%). EGFR and KRAS mutations were identified in 15% and 25% of assessable cases. None of the biomarkers was significantly associated with pts characteristics (gender, histology, smoking status), nor with RR or DCR. GART- pts experienced longer PFS when compared with the GART+ group (HR 0.56, p=.052), while no difference was observed according to TS, DHFR and AICAR status. A significantly longer OS was observed for TS- (HR 0.52, p=.012), GART- (HR 0.50, p=.037) and AICAR- (HR 0.40, p=.028) pts. No significant difference in the distribution of EGFR mutant pts receiving EGFR TKIs after pem failure was observed between IHC+ and IHC- groups. Conclusions: Low TS, GART and AICAR protein expression predicts significantly prolonged survival in single agent pem-treated pts with advanced NSCLC. The lack of PFS difference according to TS and AICAR status suggests prognostic rather than predictive relevance for these biomarkers.

Pemetrexed improves the effect of platinum-compounds against MPM, but biomarkers of response and novel effective combinations are warranted. The present study evaluated the 1) correlation between expression and polymorphisms of pemetrexed's key target TS and outcome of MPM patients treated upfront with carboplatin/pemetrexed, and 2) pharmacological interaction of new targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, and enzastaurin) with carboplatin-pemetrexed in MPM cell lines. Analysis of TS TSER-2R/3R, mRNA and protein expression was performed by PCR and immunohistochemistry (using H-score) in tumors from 99 patients. The role of TS and other molecular determinants in the interaction of carboplatin, pemetrexed with new targeted compounds was investigated in the H2052, H2452, H28 and MSTO-211H cells. Drug interaction was studied using MTT and SRB assays and evaluated with combination index method. EGFR, Akt and Erk phosphorylation, as well as VEGF secretion, were analyzed with ELISA, whereas RT-PCR and western blot were performed to assess modulation of the expression of TS, E2F-1 and genes involved in DNA repair (ERCC1 and XPD). A significant correlation between low TS protein expression and response (odd ratio, 4.2; p=0.023), longer PFS (8vs6 months; hazard ratio [HR]:0.60: p=0.023), or OS (18vs9 months; HR:0.59; p=0.029) was found when patients were categorized according to median H-score. Similarly, patients with TS mRNA below the median had longer PFS and OS (p<0.001). The higher tertile of TS mRNA expression also correlated with higher risk of progressive disease (OR:2.5; p=0.044). At multivariate analysis, TS mRNA level and H-score confirmed their independent prognostic role for PFS and OS. No correlations were observed for TSER polymorphisms. Vandetanib emerged as the targeted compound with the most potent cell growth inhibitory effects, and interacted synergistically with carboplatin and pemetrexed in all cell lines, increasing apoptotic indices. Pemetrexed enhanced EGFR phosphorylation, but reduced Akt phosphorylation and ERCC1 and XPD expression. Conversely, vandetanib significantly downregulated EGFR, Erk and Akt phosphorylation, as well as E2F-1 and TS expression (eg, H28 cells had 4.8-fold TS mRNA decrease). In conclusion, low TS protein and mRNA levels were associated to response, longer PFS and OS. Furthermore, vandetanib improved pemetrexed-carboplatin activity against MPM cells, through modulation of critical molecular mechanisms, such as EGFR-Akt phosphorylation and TS expression. These data support further prospective trials for the validation of the prognostic/predictive role of TS in patients treated with pemetrexed-based regimens, as well as future studies on the integration of vandetanib in the treatment of MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5041. doi:10.1158/1538-7445.AM2011-5041

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