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4582 Background: PI3KCA is frequently mutated in human cancer; however, information is scarce regarding its relevance in urothelial carcinoma (UC). We determined the prevalence of mutation and impact on clinical outcome of PI3KCA uniformly-treated patients with metastatic UC. Impact of PI3K and dual PI3K/mTOR inhibition was tested in vitro in UC cell lines with either H1047R or E545K mutation. Methods: 141 samples from invasive UC were scanned for mutations. Of those, complete clinical data was available from 85 cases treated with platinum-based combination chemotherapy for advanced or metastatic disease. DNA was extracted from FFPE material. Mutation status was determined by iPLEX sequencing and confirmed with hME sequencing. Overall survival (OS) was measured from beginning of treatment for metastatic disease to time of death or censored on the last known alive date. Cox proportional hazard model was used to assess the associations of PI3K mutational status and OS. Growth inhibitory effects of a specific PI3K inhibitor and a dual PI3K/mTOR inhibitor (both from Selleck) on UC cell lines with or without mutations were tested using MTT assays. Results: Mutations in the PI3KCA gene were observed in 14 (10%; 95% CI 6-16%) specimens. E545K was detected in all 14 specimens, though one specimen contained mutation at both E545K and H1047R. Among patients with clinical data, there was no statistically significant association between PI3KCA mutational status and OS (HR for having PI3KCA=0.49, 95% CI [0.15, 1.57], p-value 0.22). Preliminary in vitro experiments showed that cell growth was more potently inhibited with dual PI3K/mTOR inhibitors than with PI3K inhibitors. Conclusions: Mutations in the PI3KCA gene were detected in 10% of invasive UC and did not correlate with OS in patients with metastatic UC treated with platinum-based chemotherapy. PI3K inhibition in vitro impacts UC cell growth, though dual PI3K/mTOR inhibitors may have more significant effects than PI3K inhibition alone.

e14147 Background: In patients with metastatic colorectal cancer (mCRC), activating mutations within KRAS, which result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC have been significantly associated with lack of response to cetuximab or panitumumab therapy. Although current guidelines recommend testing for frequent KRAS codons 12/13 mutations, emerging data indicate that additional KRAS and BRAF mutations are also predictive of non-responsiveness to anti-EGFR antibodies in mCRC. This study is aimed to analyze the prevalence of low-penetrance KRAS and BRAF V600 mutations in caucasian mCRC population. Methods: A two-institution retrospective cohort of 1,238 consecutive KRAS wild type mCRC patients previously studied for 7 mutations in codons 12/13 (G12D, G12A, G12V, G12S, G12R, G12C and G13D) by the CE-IVD marked ARMS-scorpion real-time polymerase chain reaction PCR (Therascreen, Qiagen) was assayed by the diagnostic TaqMelt PCR assay cobas KRAS mutation and cobas BRAF V600 mutation tests (Roche), which are designed to detect 19 mutations in KRAS codons 12, 13 and 61 (including G12F, G13C, G13R, G13S, G13A, G13V, G13I, Q61H, Q61K, Q61R, Q61L, Q61E and Q61P) and BRAF V600 (V600E, V600K and V600D) mutations. An additional cohort of 146 KRAS mutated patients by ARMS-scorpion PCR was studied. DNA was obtained by cobas DNA preparation kit from one single 5µm formalin-fixed paraffin-embedded tissue section. Results: In all samples, sufficient DNA was obtained for KRAS and BRAF mutational studies. Among 1238 KRAS codons 12/13 wild-type patients by ARMS-scorpion PCR,166 (13.4%) showed KRAS mutations, 117 (9.5%) in codons 12/13, and 49 (4%) in codon 61. BRAF V600 mutations were detected in 9% cases. In ARMS-scorpion PCR KRAS mutated patients, mutations were confirmed by cobas in all cases. Conclusions: The cobas mutation tests are robust and reproducible assays that, 1) detects a higher incidence (13.4%) of mutations in codons 12, 13, and 61 of the KRAS gene in wild-type mCRC population, 2) a relevant rate of BRAF mutations is present in the same population, and 3) requires a very small amount of tissue.

e15094 Background: Activation of VEGFR through its phosphorylation at specific tyrosine residues promotes changes in the microvasculature density. Inhibition of this mechanism targeting kinase activity has demonstrated an impact on clinical benefit in renal cell carcinoma. Methods: Fifty renal cell carcinomas have been assessed to evaluate the role of VEGF-VEGFR pathway and its impact on microvascular density (MVD). In order to identify parameters of potential clinical benefit to sunitinib a control group was included. IHC of VEGF, VEGFR (KDR), phosphorylated VEGFR-Y1175 and microvascular density assessment were performed. Results: All cases show VEGF expression; using a ROC curve a threshold was identified with a statistical significant worse PFS in those cases with higher VEGF expression in the whole series of patients treated with sunitinib (p=0.037) and in the subgroup with clear cell histology only (p=0.011). pKDR was positive in 40%: with an AUC=0.9 and according to this threshold statistical significance was found in PFS in the whole series (p=0.016) and in the clear cell subgroup (p=0.007). Microvascular density showed a statistical significance when was evaluated in terms of PFS in the group including all histologies (p=0.011) and those cases with clear cell subtype only (p=0.005). Conclusions: Our data suggest that VEGF, pKDR-Y1175 and MVD may identify the patients with significant clinical benefit to sunitinib in terms of statistically longer PFS.

4580 Background: Incidence of Her2 positivity and association with overall survival (OS) are controversial in advanced UC. Activating Her2 mutations have been identified in other cancers, but they have not been previously reported in UC. We determined Her2 status by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and copy number gain (CNG) via array CGH of primary UC tumors from patients (pts) with metastatic disease. Targeted Her2 sequencing was performed at known mutation hotspots, and mutation effect was investigated in vitro. Methods: Tissue microarrays of formalin fixed paraffin-embedded tumor from 98 UC pts treated with platinum-based combination chemotherapy for metastatic disease were evaluated for Her2 protein and for Her2 gene amplification by using standard clinical protocols. Positive staining was defined as an IHC score of 3+ or a FISH ratio of ≥2 using scoring criteria established for evaluation of breast cancer. Her2 CNG was evaluated by aCGH with cutoff log base 2 ratio > 0.9. Mutation status was validated by hME sequencing. OS was measured from start of treatment for metastatic disease. Association of OS and Her2 status was assessed by a Cox regression model. NIH-3T3 cells with Her2 V777L were assessed for growth, invasion, and Her2 kinase activation. Results: 22% of pts had 3+ Her2 staining by IHC. 21% of pts had FISH amplification. These were concordant in 78% of pts. CNG was identified in 16% and was concordant with FISH and IHC 85% and 88% of the time, respectively. Her2 status by any modality showed no significant association with OS in either univariate [HR=0.94, 95% CI: (0.52, 1.70), p=0.83] or multivariate [HR=1.12, 95% CI: (0.61, 2.06), p=0.72] analysis. Her2 mutations (V777L and L755S) were identified in 2 pts (2%). In vitro analysis of V777L results in transformation of NIH-3T3 cells, leading to increased growth, invasion on soft agar, and Her2 kinase constitutive activation. Conclusions: Her2 overexpression or amplification in the primary tumor does not predict OS in pts with metastatic UC. Other research has suggested that V777L sensitizes cells to lapatinib, while L755S leads to lapatinib resistance. These rare oncogenic Her2 mutations occur and may be therapeutic targets in selected pts.

e18106 Background: Mutations in the Epidermal Growth Factor Receptor (EGFR) predict a better outcome to EGFR tyrosine kinase inhibitors than platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC). Previous studies with Caucasian patients have shown a prevalence of EGFR mutation of 10-15%. The aim of this study is to analyze the prevalence of EGFR mutations in squamous-cell NSCLC patients from an area of influence of 500,000 habitants. Methods: Prospective mutation testing in NSCLC patients included in our institution since October 2010 to January 2012 was performed on DNA obtained from available tumor tissue and cytologic samples, using ARMS-scorpion TheraScreen EGFR 29 Mutation Test Kit (Qiagen). Results: From 218 consecutive NSCLC diagnoses, 18 (8.25%) patients showed EGFR mutations: 6 (33.3%) exon 19 deletion, 9 (50%) exon 21 mutations (7 L858R and 2 L861Q) and 3 (16.6%) cases exon 20 insertion. In the EGFR mutated population, 16 (88.88%) patients were diagnosed as adenocarcinoma and 2 (11.11%) as squamous cell carcinoma. The characteristics of these squamous cell cancer patients were: 2 male; 1 non-smoker, 1 former-smoker; 1 stage IV and 1 stage IB at diagnosis; one case exon 20 insertion and one exon 21 mutation (L858R). Conclusions: The EGFR mutation rate in squamous-cell NSCLC patients in our referral area is superior (11.17%) than previously reported, reinforcing the importance of including EGFR mutation testing in squamous-cell NSCLC population for selecting optimal therapy for these patients.

10616 Background: Ki67 proliferation biomarker determined by immunohistochemistry (IHC) has been studied as a prognostic and predictive factor in Operable Breast Cancer (OBC). Ki67 modifications after neoadjuvant endocrine therapy have been correlated with long term outcome. However, there is no robust data about its predictive role in Neoadjuvant Chemotherapy (NC). In this study, we investigated Ki67 value as predictor of NC efficacy. Methods: 193 patients (pts) from 2 GEICAM phase II randomized trials (2006-03 and 2006-14) were included: 78 (40%) received epirubicine plus cyclophosphamide followed by docetaxel (EC-D), 41 (21%) EC-D plus carboplatin, and out of the 74 HER2+ pts, 37 (19%) received EC-D plus tratuzumab and 37 (19%) EC-D plus lapatinib. Median age was 49 years. From series, 87% were invasive ductal carcinoma, 58% premenopausal, 50% grade III, 23% luminal , 39% basal and 38% HER2+. Ki67 was centrally assessed by IHC (MIB1 clone) and median score was 40% (range 1-100%). Pathological Complete Response (pCR), defined as absence of invasive cells in breast and lymph nodes, was achieved in 56 pts (29%). Univariate and multivariate logistic regression models were used to study the association of each clinical-pathological variable with pCR. ROC curves were used to determine the most accurate ki67 cut-off for predicting NC response. Results: Ki67≥50% was defined as the most accurate threshold to select patients obtaining benefit from NC. In the univariate analysis, histological grade (p=0.01), treatment (P=0.006), ER (p

4585 Background: Advanced UC is associated with multiple SNCAs. To identify SCNA predictors of poor survival in advanced UC, we evaluated SCNAs in two cohorts of high-risk urothelial carcinoma of the bladder. Methods: We obtained DNA copy number data from bladder cancer patients in two independent cohorts [Spanish cohort (n= 93, Agilent aCGH 180k array) and Brigham and Women’s/Dana-Farber (BW/DF) cohort (n = 48, Affymetrix OncoScan FFPE Express 2.0)]. For the BW/DF cohort, we acquired copy number information from 30 primary tumors and 33 metastases. For 12 patients, both primary and matched metastases were available. The GISTIC method was used on GLAD segmented data to identify driver copy number lesions, and the NBC algorithm to identify recurrent breakpoints. Cox proportional hazards models were used to estimate the effect of the identified SCNAs on overall survival, defined as time from start of chemotherapy for metastatic disease (Spanish) or from metastatic recurrence (BW/DF). Copy numbers were incorporated in the Cox models as continuous measures. Statistical significance of optimal cutpoints was estimated with permutation tests. Copy number profiles of primary tumors and metastases were compared with hierarchical clustering. Results: Amplification of 1q23.3 was independently associated with overall survival in both cohorts (Spanish cohort: HR 3.98; 95% 1.64-9.67; p < 0.03; C-statistic 0.54, 95% 0.45-0.65. BW/DF cohort: HR 6.28; 95% 1.84-21.4; p < 0.042; C-statistic 0.62, 95% 0.48-0.76). Amplifications at 22q12.2 were enriched in patients who developed metastasis (p < 0.05). A breakpoint analysis identified possible truncations of ITPR1 (Spanish: 15%; BW/DF: 25%) and PRIM2 (Spanish: 31% BW/DF: 12.5%) in large numbers of samples. For primary tumors with matched metastases, we observed that metastases clustered together with their primary tumors. Conclusions: External validation of the 1q23.3 amplification demonstrates the association of this SCNA with poor outcomes in advanced UC; the identification of the target of this copy number gain is ongoing. Gene truncations and their biological significance require further experimental investigation.

e18069 Background: KRAS mutation are harbored in ~30% of NSCLC patients, and have been described associated with an upregulation of genes involved in angiogenesis, including vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Bevacizumab (BVZ) is a recombinant monoclonal humanized antibody targeted against VEGF and added to carboplatin-paclitaxel chemotherapy has demonstrated to improve outcome in non-squamous NSCLC. The objectives of this study were to analyze the impact of KRAS mutations in response rate (RR) and time to progression (TTP) in a cohort of patients homogeneously treated with BVZ in combination with carboplatin-docetaxel. Methods: Patients treated with up to 6 cycles of carboplatin (AUC 5), docetaxel (75 mg/m2) and BVZ (7.5 mg/kg) on day 1, every 21 days. Patients with RR or stable disease continued maintenance BVZ (7.5 mg/kg) every 21 days until disease progression. KRAS mutations at codons 12, 13 and 61 were analyzed by COBAS KRAS Mutation Test (Roche) from DNA purified from diagnostic samples. Results: 30 patients were enrolled (24 male, 6 female); median age was 62 years (range 44-82); ECOG 1/2: 22/8. 27 were adenocarcinomas and 3, undifferentiated large cell carcinoma. Smoking habit: 1 non-smoker, 16 current-smoker, and 13 former-smoker. Seven patients were KRAS mutated (23.3%). Carboplatin-docetaxel-bevacizumab therapy showed a significant improved efficacy in KRAS wild-type compared to mutated patients (RR: 85.7% vs 14.3%, p=0.045; and TTP: 9.38 months vs 7.46 months, p=0.033), but did not impact on overall survival (12.4 months in wild type vs 11.06 months in KRAS mutated, p= 0.780). Conclusions: KRAS mutations predict RR and TTP in wild-type EGFR stage IV non-squamous NSCLC patients treated with bevacizumab-based therapy.

5520 Background: Activation of the c-MET oncogene promotes tumor growth, invasion and metastasis in several tumor types. In addition, c-MET has been reported in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade resulting a mechanism of acquired resistance to EGFR inhibitors in lung and colorectal carcinoma. We investigated the impact on cetuximab sensitivity of HGF, c-MET, c-MET activation and c-MET gene status in recurrent or metastatic squamous cell carcinoma of the head and neck (HNSCC) patients. Methods: A single-institution retrospective analysis including 50 HNSCC patients was carried out. For each case, formalin-fixed tumor specimens were assayed for HGF, total and phosphorylated Y1234/35 c-MET (p-c-MET) by immunohistochemistry and c-MET gene by FISH. Overexpression criteria were defined by ROC curves for each protein and amplification was defined by >2 copies in at least two of the three studied tumor areas. Results were analyzed for association with clinico-pathological features and survival outcomes for cetuximab-treated patients (median follow-up 75 months). Results: c-MET overexpression was detected in 26 (52%) of patients, c-MET amplification in 15 (30%) and p-c-MET in 12 (24%). Amplification was associated with c-MET overexpression (p=0.004). HGF overexpression was observed in 8 (12%) associated with c-MET phosphorylation (p=0.001), suggesting a paracrine activation of receptor. No significant association with clinico-pathological parameters was detected. Log-rank test showed significantly worse outcome in c-MET overexpressing patients for progression-free (PFS) (p=0.002) and overall survival (OS) (p=0.045); p-c-MET was correlated with worse PFS (p=0.014) and OS (p

e21015 Background: Mutations in the Epidermal Growth Factor Receptor (EGFR) predict a better response to tyrosine kinase inhibitors compared to platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC). Previous studies in caucasian population have reported a frequency of EGFR mutation of 10-15%. The aim of this study is to analyze the prevalence of EGFR mutations in caucasian smoker patients with NSCLC. Methods: Prospective mutation testing in NSCLC patients included in our institution since October 2010 to January 2012 was performed on DNA obtained from available tumor tissue and cytologic samples, using ARMS-scorpion TheraScreen EGFR 29 Mutation Test Kit (Qiagen). Results: From 218 consecutive NSCLC diagnoses, 18 (8.25%) patients showed EGFR mutations: 6 (33.3%) exon 19 deletions, 9 (50%) exon 21 mutations (7 cases L858R and 2 cases L861Q) and 3 (16.6%) exon 20 insertions. In the EGFR-mutated patients, 13 (72.25%) were never-smoker and 5 (27.7%) were smoker (3 current-smoker and 2 former-smoker). The EGFR-mutated smoker population showed the following characteristics: 3 female, 2 male; 4 adenocarcinoma, one squamous cell carcinoma; 3 stage IV, 2 stage IA at diagnosis; 1 (20%) case EGFR deletion exon 19, 1 (20%) case insertion exon 20 and 3 patients (60%) mutation exon 21 (2, L858R and 1, L861Q). Conclusions: The EGFR mutation rate in NSCLC smoking patients in our referral area is superior (27.7%) than previously reported, reinforcing the importance of including EGFR mutation testing in NSCLC smoking population for the correct management of these patients.

Dual-specificity phosphatase type 1 (DUSP1/MKP1), is a member of the dual-specific family of phosphatases that can dephosphorylate and inactivate all three major Mitogen-Activated Protein Kinases (MAPKs), including Extracellular Regulated Kinase (ERK1/2), c-Jun N-Terminal Kinase (JNK1/2), and p38. MKP1 is a nuclear protein whose expression is regulated by mitogenic, inflammatory and DNA-damage stimuli. Basal levels of MKP1 are usually low in normal cells, however high levels of the protein have been found in several human tumours such as ovarian, breast, prostate and lung. Amplification and mutation of Ha-Ras correlates with the malignancy of tumours that appears in chemically-induced mouse skin and additionally, an increase in Ha-Ras levels induces JNK1/2 and ERK1/2 activity, known substrates of MKP1, however, there is no evidence in the literature of a relationship between MKP1 and skin cancer. The objective of this study was to investigate the role of MKP1 in carcinogenesis and tumor development in carcinogen-induced skin cancer. MKP1 wild-type (mkp1+/+) and MKP1 knock-out (mkp1-/-) mice were subjected to the standard two-stage skin carcinogenesis protocol using 7, 12-dimethylbenz(a)anthraacene (DMBA) as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. We observed a significant decrease in both papilloma incidence and papilloma multiplicity in mkp1+/+ mice undergoing two-stage skin carcinogenesis relative to mkp1-/- littermates. Morphological evaluation of skin lesions from mkp1+/+ and mkp1-/- mice revealed a significant reduction in both tumour incidence and tumour multiplicity in mkp1+/+ mice relative to mkp1-/- littermates. No differences were found between mkp1+/+ and mkp1-/- skin hyperplasia following TPA challenge, but we observed reduced bromodeoxyuridine (BrdUrd) incorporation in mkp1+/+ epidermis compared with mkp1-/- epidermis. Targeted disruption of MKP1 gene led to a slight increase in ERK1/2 protein in mkp1-/- mice, whereas p38 and JNK1/2 proteins were expressed at similar levels in the epidermis of mkp1+/+ and mkp1-/- mice. We examined the effect of MKP1 deficiency on ERK1/2 activation and we observed that TPA-mediated activation of ERK1/2 was diminished in mkp1+/+ epidermis compared with mkp1-/- epidermis. Finally, we have also observed a marked reduction in MKP1 expression levels in human basal (BCC) and squamous cell carcinoma (SCC) in relation to normal skin. In contrast to the results found in other tumor types as lung cancer, our data provide a strong evidence for MKP1 having a role in the tumorogenesis and development of skin cancer by attenuating epidermal response to tumour promotion, and ultimately, two-stage skin carcinogenesis, very likely by impairing the activation of the ERK1/2 pathway, critically linked to control of cell proliferation and skin tumorigenesis. Supported by PI081485 and P09/1988 and by an unrestricted educational grant from Fundación Mutua Madrileña 2007-0444 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2366. doi:1538-7445.AM2012-2366

291 Background: DNA repair factors are hypothesized to mediate chemosensitivity to cytotoxic agents that produce DNA damage and may be predictive for response to platinum-based chemotherapeutic regimens. Primary urothelial carcinoma (UC) tumors of patients who developed metastatic disease were evaluated for expression of a panel of DNA repair factors by immunohistochemistry (IHC). Methods: A cohort of 132 clinically annotated, uniformly-treated (platinum-based combination chemotherapy) UC patients who subsequently developed distant metastases with FFPE primary tumor tissue available was identified. Tumor bearing areas were evaluated by a single urologic pathologist. Tissue arrays were constructed for IHC analysis of the following DNA repair factors: ERCC1, Rad 51, BRCA1/2, PAR and PARP1. Tumor was deparaffinized and specific antigen retrieval determined for individual antibodies. Pathologist supervised IHC analysis of nuclear versus cytoplasmic expression was performed using spectral imaging analysis. Overall survival (OS) was defined from start of chemotherapy for metastatic disease to death (N=67) or censored on the last known alive date. Percent of positive nuclear staining was categorized as quartiles or previously identified cut-points. Cox regression evaluated the associations of percent positive nuclear staining levels and OS in multivariable analysis (HRs and 95% CIs included) that controlled for known prognostic variables (performance status and visceral metastases). Results: Higher percentage of nuclear staining of ERCC1 [HR=2.7 (1.5, 4.9), p=0.0007]; Rad51 [HR=5.6 (1.7, 18.3), p=0.005]; and PAR [HR=2.2 (1.1, 4.4), p=0.026] in tumor tissue were associated with poor outcome; each was independent of the other two repair factors and known prognostic variables. Neither nuclear nor cytoplasmic staining for BRCA1/2 or PARP1 reached statistical significance for an association with OS. Conclusions: DNA damage repair factor levels measured by IHC impact outcomes in advanced UC. External validation is ongoing and will be presented. Further studies are required to determine whether these biomarkers are prognostic or predictive in this setting.

Background: The addition of trastuzumab (T) to neoadjuvant chemotherapy increases pCR rate in patients with HER2 + breast cancer. Lapatinib (L) in combination with chemotherapy is also an active drug in breast cancer patients. This study investigates the efficacy of trastuzumab or lapatinib added to chemotherapy in the neoadjuvant setting. Methods: Patients (Pt) with tumors greater than 2 cm (or less with positive axilla) and HER2+ (Herceptest 3+ or 2+ with FISH+) that have not received any prior treatment for breast cancer were recruited. Pts were randomized to receive epirubicin 90 mg/m2 plus cyclophosphamide 600 mg/m2 × 4 cycles (cy) followed by docetaxel 100 mg/m2 (with growth colony stimulating factor support) plus either T 8 mg/kg loading dose and then 6 mg/Kg intravenous (EC-DT) or L 1250 mg orally (EC-DL). Patients were stratified according to tumor size (T1-2 vs T3-4), and estrogen receptor status (positive vs negative). The primary end-point was pathological complete response (pCR) in the breast measured by Miller and Payne criteria. Secondary end-points were clinical response (by imaging test), toxicity and correlation between pCR and tumor biomarkers. Results: From February-09 to October-10, 102 pts were randomized (50 EC-DT, 52 EC-DL). Pts characteristics were well balanced between arms. Median age was 48 years (30-79), 56% of pts were premenopausal, 7% grade I, 45% grade III. 14/59/12/15% were T1/T2/T3/T4 and 69% N+. 58% were estrogen receptor positive and 44% progesterone receptor positive. Three patients were FISH negative after central assessment and were not considered evaluable for efficacy. pCR rate in the breast was 52% (95% CI: 38–66) for EC-DT and 25% (95% CI: 13.5−37.5) for EC-DL (p-value=0.0065). pCR—pN0 was 48% (95% CI: 34–62) for EC-DT and 24% (95% CI: 12 - 35) for EC-DL (p-value=0.01). Clinical response was 77% with EC-DT and 65% with EC-DL (p-value=0.176). Grade III-IV toxicity was similar between arms except for diarrhea that was more frequent in EC-DL 14% than in EC-DT 2% (p-value=0.03); more patients discontinue treatment due to toxicity with EC-DL (1 vs 6; p-value=0.055). Conclusions: Our study shows that EC-DT was more efficacious and less toxic than EC-DL in the neoadjuvant treatment of HER2+ pts. Biomarker profiling in the tumors, including activation levels of tyrosine-kinase receptors, signaling pathways and surrogate markers (proliferation and apoptosis) is ongoing; their correlation with pCR will be presented at the meeting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD07-04.

4569 Background: Limited information exists regarding the changes that occur in UC tumors that ultimately metastasize, the lethal phenotype of UC. Recurrent copy number alterations (CNAs) have been...

10585 Background: Neuroendocrine tumors (NETs) comprise a heterogeneous group of neoplasms containing biologically active peptides and amines in their cytoplasm. Anaplastic lymphoma kinase (ALK) tr...

The identification of oncogenic chromosomal alterations of anaplastic lymphoma kinase (ALK) in lung cancer and neuroblastoma has revealed ALK as a potential candidate for anticancer targeted therapy. Thus, ongoing clinical trials with ALK inhibitors are showing promising results. ALK chromosomal alterations in other solid tumors are controversial. The aim of this study was to analyze ALK translocations and gene copy number status in a series of colorectal cancer patients and correlate these findings with tumor pathological features including TNM staging, KRAS and BRAF mutations, as well as clinical outcome. ALK status was retrospectively evaluated by fluorescence in situ hybridization (FISH) with a break-apart ALK (2p23) probe (Abbott Molecular, Des Plaines, IL, USA) and centromeric probe of chromosome 2 (Abbott Molecular) in biopsies from colorectal cancer primary tumors, as were KRAS and BRAF by mutational analysis. Survival was analyzed by KaplaneMeier method. One hundred and four colorectal cancer patients were evaluated. ALK translocations were not observed; however, 39/104 (37%) of the patients had an increase in ALK gene copy number (3e5 copies), with the percentage of cells with ALK copy gain within a tumor ranging between 6% and 46% (mean 22%). There was no correlation between ALK gene copy number and KRAS mutational status, but BRAF mutations and ALK gain appear to be mutually exclusive, since the five patients with BRAF mutation had ALK disomic pattern. ALK gain was correlated with nodal status. Forty-three per cent of patients with positive nodal status had an increase in ALK copy number, while only 15% of patients with negative lymph nodes had ALK gain (p 5 0.05). The increase in ALK gene copy number was not correlated with overall survival. As a conclusion, the identification of ALK gene gain in colorectal cancer patients seems to be a relatively frequent genetic abnormality and is associated with tumors that have a more aggressive phenotype. Taking into account that ALK inhibitors are showing promising results in other types of tumors, it could be interesting to study ALK as a potential new therapeutic target for this tumor. Further evaluation of the biological meaning of ALK gain in colorectal cancer is ongoing. GERMLINE EPIMUTATION OF THE TUMOR SUPPRESSOR GENE PTPRJ IN EARLY ONSET FAMILIAL COLORECTAL CANCER

Trabajo presentado al 21th Meeting of the European Association for Cancer Research celebrado en Bruselas (Belgica) del 25 de Junio al 30 de Junio de 2010.

e14018 Background: The validation of KRAS mutations as a negative marker of response to the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab has meant a seminal advance towards treatment individualization of colorectal cancer (CRC) patients. However, since KRAS wild-type status does not guarantee response to anti-EGFR antibodies, identification of other biomarkers of response is urgently needed. We hypothesized that MKP-1, a phosphatase that inactivates phospho-MAPKs including the EGFR downstream protein ERK, could be a mediator of resistance to anti-EGFR antibodies. Methods: Tumor specimens from 48 metastatic CRC patients treated with cetuximab-based chemotherapy were evaluated for KRAS and BRAF mutational status as well as MKP-1 expression as assessed by immunohistochemistry. Results: As expected, clinical benefit was confined to wild-type KRAS and BRAF patients. MKP-1 was overexpressed in 16 patients (33%) and was not associated with patient baseline clinical characteri...

611 Background: NF-kB is a potential therapeutic target in breast cancer and other tumor types. However, studies assessing its significance in a clinical setting are as yet limited. The aim of this study was to evaluate clinicopathological associations and predictive value of the transcription factor NF-kB in a large series of breast cancer patients that received neoadjuvant systemic treatment. Methods: A retrospective search of a prospectively maintained database was performed to identify patients. Immunohistochemistry was employed to asses the p65 subunit of NF-kB using nuclear staining, as a surrogate of activation. Results: Nuclear NF-kB expression (n = 133) was found in 26.3% (n = 35) of cases. Nuclear NF-kB staining was associated with high histological grade (p = 0.05), ER negativity (p = 0.01) and high Ki67 index (p = 0.002). Patients with nuclear NF-kB staining had a higher pathological complete response (pCR) rate compared to those without (26.5% vs. 6.0% respectively, p = 0.004); there were no ...

10537 Background: The identification of oncogenic chromosomal alterations in anaplastic lymphoma kinase (ALK) in lung cancer and neuroblastoma has revealed ALK as a potential candidate for anticanc...

e14617 Purpose: HGF/MET pathway is aberrantly activated by receptor overexpression and mutations in SCLC preclinical models, enhancing their oncogenicity. The significance of MET expression in SCLC remains unclear. Our aim was to analyze the effects of MET inhibition in chemosensitive/refractory SCLC models and to study the expression pattern and prognostic impact of total and phosphorylated (p) MET in SCLC patients. Methods: Total and p-MET expression (Western Blot), gene copy number (FISH), and exon 14 activating mutations (sequencing) were evaluated in H69 and H69AR SCLC cell lines. PHA-665752 (PHA), alone or combined with doxorubicin, was used to study the effects of pathway inhibition on viability, colony formation and invasion assays in basal/stimulated conditions (HGF). Fifty-eight SCLC cases were evaluated for MET and p-MET expression by immunohistochemistry. Survival analyses were performed. Results: H69 and H69AR (both R988C mutated) expressed MET at basal conditions, but not p-MET. HGF induced MET phosphorylation, increased proliferation (20%) and protected cells from doxorubicin cytotoxicity. PHA 0.5μM blocked MET phosphorylation, decreased colony formation by 50% in H69, counteracted the cytoprotective effect of HGF and inhibited invasion in H69AR. MET expression was found in 98% normal bronchial epithelia, and 78% tumor samples (overexpression 38%). Activated MET was focally detected in normal and metaplastic mucosa and expressed in 22% tumors. MET expression was associated with improved overall and disease free survival (p: 0.06 and 0.051, respectively). All p-MET positive cases within MET expressing tumors, showed relapsed disease (83% in negative p-MET samples, p=0.065), suggesting MET activation may revert the good prognosis linked to total MET expression. Conclusions: MET activation, results in a more aggressive phenotype in SCLC cells. PHA at MET inhibiting concentrations reverses this phenotype. In SCLC specimens, MET expression was more prevalent than p-MET and associated with raised prognosis. All these data suggest that studies with MET inhibitors should focus on p-MET positive SCLC. [Table: see text]

5025 Background: DNA damaging agents are the backbone of combination chemotherapy regimens for the treatment of advanced bladder cancer. We hypothesized that levels of DNA repair genes such as ERCC1 could predict survival in patients receiving platinum based therapy. Our previous work (Bellmunt J et al, Ann Oncol. 2007) showed that survival was significantly higher in advanced bladder cancer patients with low gene expression levels of ERCC1 measured by RT-qPCR (25.4 versus 15.4 months; p = 0.03). We aimed to confirm these findings using immunohistochemistry (IHC) in an independent cohort of advanced bladder cancer patients treated with cisplatin-based chemotherapy at our institution. Methods: Formalin-fixed paraffin-embedded tumor tissue was available from 51 patients. IHC stains for ERCC1 protein levels were scored as percentage and intensity of positive cells. ERCC1 staining was considered positive if detected in ≥1% tumor cells. The Kaplan-Meier method was used to calculate survival and the Cox proportional hazards model was used to examine the prognostic value of protein expression levels. Results: ERCC1 staining was negative in 60% of patients, weakly positive (1–10% positive cells) in 21% and strongly positive (>10% positive cells) in 19%. Median overall survival for all patients was 14.4m (95% CI 6.7–16.1m). Median disease-specific survival was significantly higher in patients negative for ERCC1 by IHC (12.6m versus 8.6m; p = 0.032). Conclusions: Our results using the more simple IHC technique confirm the prognostic value of ERCC1 expression in advanced bladder cancer. Patients with high expression of ERCC1 by IHC have a worse disease-specific survival than patients with no expression. A correlative study of IHC and RT-qPCR in both sets of samples is ongoing. No significant financial relationships to disclose.

6070 Background: Nimotuzumab (N) is a humanized MAb to EGFR that has shown evidence of efficacy in patients (pts) with advanced solid tumors, notably without provoking skin rash. Although lack of r...

4065 Background: AEE788 (AEE) is an oral inhibitor with potent activity against EGFR, ErbB2, and KDR. This phase I study was to assess the safety, PK, PD, and MTD/DLT of AEE in pts with CRC and liver metastases. Methods: AEE was given PO at 25, 50, 100, 250, 300 or 400 mg/day daily in 28-day cycles (C) to 3–6 pt cohorts. 24-hr PK was obtained on C1, days (D)1, 15 and 28. PK parameters of AEE and active metabolite, AQM674 (AQM) were computed by non-compartmental methods. PD markers were analyzed in skin (SK), and tumor (TU) biopsies pre- and post-treatment. Samples were evaluated by both IHC and Laser Scanning Cytometry (LSC). DCE-MRI was performed at baseline (BL), and on C1D2 and 28 and C2D28. Results: 22 pts were treated at doses of 25 (n=4), 50 (n=3), 100 (n=4), 250 (n=1), 300 (n=4), and 400 mg (n=6). No DLT was reported. The most common AE (> 15%): fatigue (55%), vomiting (46%), diarrhea (41%), nausea (36%), dyspnoea (23%), constipation (18%) pyrexia (18%) and rash (18%). 2 pts had reversible gr 3/4 LFTs. Serum concentration of AEE and AQM increases with dose and dose duration. 7 pts had stable disease at the end of C2. Median time on treatment was 56 days (range 7–168). In SK, at doses =100 mg, pEGFR, pMAPK and Ki67 were inhibited by an average of 51, 54 and 41%, respectively by IHC. In endothelial cell (EC) pKDR/KDR was significantly inhibited (4-fold) in the wound-induced SK in a dose-dependent manner by LSC. In TU, inhibition of pEGFR, pMAPK and Ki67 at 400 mg was 100%, 90% and 39%, respectively by IHC. EC pKDR decreased with dose, however, trend was not significant. 1 out 15 pts who had BL and end of C1 DCE-MRI had > 40% Ktrans reduction. Conclusions: A dose dependent inhibition of pKDR, pEGFR, and pMAPK in skin and tumor was observed. However, no significant effects were observed on Ki67 and apoptosis in TU. No dose dependent reduction in Ktrans from DCE-MRI was observed. These findings were consistent with the lack of clinical activity of AEE up to 400 mg in this patient population. The study has been discontinued without reaching DLT due to safety data from other phase I studies and in favor of different dosing schedules. No significant financial relationships to disclose.

3511 Background: The PI3K/Akt/mTOR pathway is found dysregulated in multiple tumours, including breast cancer (BC). RAD001 is a recently developed drug which blocks mTOR. To study this compound’s anti-tumour effect and interactions with the aforementioned pathway, we set up a translational research program which integrates basic and clinical research data. Methods: The preclinal studies performed in BC cell lines and xenografts included the use of proliferation assays, western blot (WB), small interfering RNA and immunohistochemistry (IHC) analysis. The clinical studies consisted in a phase I study with escalating doses and collection of tumour biospies at different time points Results: Our first achievement was validating the antibodies used in WB and IHC and directed towards different components of the PI3K/Akt/mTOR pathway. We first reported that sensitivity to RAD001 correlated to basal levels of p-Akt. We then observed that RAD001 inhibits mTOR downstream, pS6 and eIF4G, but increases the levels of p-Akt, both in vitro and in patients’tumour biopsies. We demonstrated that knock down of the pS6 kinase mimics the effect of RAD001. Indeed, by combining the PI3K inhibitors and the genomic approch of p85 dominant negative vector, we demonstrated that RAD001 induced p-Akt is through PI3K. Besides the p-Akt induction we also found a RAD001 induction of IRS-1, the pivot mediator of the Insulin like Growth factor-1 Receptor (IGF-1R). We reported that RAD001 resulted in a prolonged activation of IRS-1 and in a sustained association with the p85 subunit of PI3K. Thus RAD001 disrupts an inhibitory mechanism impinging on the PI3K signalling, resulting in sustained activation of the IGF-1R pathway. Accordingly we demonstrated that combination of RAD001 with coumpounds directed against the IGF-1R, i.e. both IGF-1R tyrosine kinase inhibitors and monoclonal antibodies, prevented RAD001 induced p-Akt and resulted in a supradditive growth inhibitory effects both in vitro and in MCF-7 human BC derived xenografts.We are now conducting a phase I study with a monoclonal antibody directed at the IGF-1R that blocks IGF-1R signalling. Conclusions: Dual inhibition of mTOR and IGF-1R completely blocks the PI3K/Akt/mTOR pathway and provide a mechanistic-based combined approach. Clinical studies in BC patients are being planned. No significant financial relationships to disclose.

3006 Background: C and G are two anti-EGFR agents with different mechanisms of action. We had previously shown a synergistic effect combining the two agents in preclinical models (Matar et al, Clin Cancer Res 2004). This study aims to explore safety, PK and PD changes in tumor and skin at different doses of C/G to define the recommended dose (RD) for further development. Methods: pts were treated at the RD of weekly iv C (400 mg/m2 initial dose, 250 mg/m2 weekly) and oral daily G (250 mg/d) as single agents (5 pts each) and in successive cohorts of combined C/G (3–6 pts each): C (320/200) / G (100), C (400/250) / G (100), C (400/250) / G (250), C (320/200) / G (500) & C (400/250) / G (500) (ongoing). Dose escalation depended on dose limiting toxicity (DLT) rate during the first 28 d. Pre- & on-treatment steady-state (14 d) tumor & skin biopsies were obtained and have been evaluated by IHC for total (t) and phospho (p)-EGFR, p-MAPK, p-Akt, proliferation (Ki67), p27 expression and apoptosis by TUNEL. Gene profiling analysis is ongoing. Results: 35 pts have been treated so far: 20 CRC, 13 HNC & 2 NSCLC; median KI 90 % (70–100); median age 60 years (38–80); 24 males, 11 females. DLTs occurred in 3 pts: 1 pt with G alone with reversible ILD; 1 pt with G (250) / C (400/250) with reversible deafness & 1 pt with G (500) / C (320/200) with grade 3 anorexia and nausea. There were 1 CR (HNC) and 5 PRs (CRC) in the C/G cohorts, and 1 PR (CRC) in the C cohort. Overall, 5 out of 9 (56%) pts with CRC treated in the C/G cohorts presented a PR. PD studies show superior inhibition of p-EGFR, p-MAPK and p-Akt, reduction of proliferation and increased apoptosis (all p values [Table: see text]

7160 Background: The HER1/EGFR inhibitor erlotinib significantly prolongs survival of patients with previously-treated advanced NSCLC. Methods for selecting patients most likely to derive clinical benefit from erlotinib are not established. Increased HER1/EGFR gene copy number has been suggested as a potential predictive biomarker of clinical benefit, and was investigated in this phase II study. Methods: Advanced NSCLC patients who failed first line chemotherapy were treated with erlotinib monotherapy, 150 mg/d p.o. Each patient underwent tumor biopsy before start of treatment. Tumor HER1/EGFR gene amplification status was assessed using FISH, and classified as positive (amplification, polysomy, high polysomy) or negative (disomy, trisomy). Results: 83 patients were included: median age 56 (range 35–78); sex: male 72%, female 28%; histology: adenocarcinoma 43%, large cell 31%, squamous cell 19%, others 7%; smoking status: 44 current smokers, 28 former smokers, 11 never smokers. Of 73 evaluable patients, 7 (10%) achieved partial response (PR), 28 (38%) had stable disease (SD) and 38 (52%) had disease progression. PRs were observed in 4 males / 3 females; in 5 adenocarcinomas / 1 large cell/ 1 squamous cell; in 2 current / 3 former / 2 never smokers. Erlotinib was well tolerated and no unexpected toxicities were seen. HER1/EGFR gene copy number was evaluated in 53 patients. 15 patients were FISH +, 10 of whom achieved clinical benefit (PR, or SD for ≥12 weeks). Only 5 of 38 FISH - patients had clinical benefit. FISH + patients achieved a longer median time to progression (137 vs 43 days; p = 0.00011; HR 0.35) as well as overall survival (226 vs 115 days; p = 0.3221, HR 0.722). Conclusion: In this study, increased HER1/EGFR gene copy number was associated with a better outcome on erlotinib therapy. [Table: see text] [Table: see text]

3085 Background: The anti-EGFR Mab C is active in mCRC. Since C has a long terminal half-life and it is frequently given in combination with q2w chemotherapy, we decided to compare the safety and PK/PD of a more convenient q2w schedule with the currently approved q1w schedule in pts with EGFR-expressing mCRC. Methods: In this study C was given q2w at the standard q1w regimen (400 mg/m2 initial dose and 250 mg/m2 weekly) to 10 pts as a comparator. In the experimental arm, C was given q2w with a dose escalation in sequential steps for cohorts of 10 pts each (400, 500, 600 & 700 mg/m2). Dose levels were escalated if no dose-limiting toxicities (DLTs) were observed. DLTs were defined as grade 3–4 toxicities (except for hypersensitivity reactions and grade 3 skin toxicity), and/or administration of less than 66% of the assigned dose due to toxicity. In order to conduct the PK/PD studies, all treatment groups were given C alone for 6 weeks and subsequently, the FOLFIRI regimen was added. Complete PK profile was obtained during the initial 6 weeks of treatment. Skin and tumor biopsies for PD profiling were obtained prior to therapy and on day 28 (steady state) and have been analyzed by IHC for total (t) and phospho (p)-EGFR, p-MAPK, p-Akt, proliferation (Ki67) & p27 expression. Results: 29 pts have been included so far: 9 in the q1w group and 20 in the q2w group (10 pts each at 400 and 500 mg/m2). To date there have been no DLTs. PK data are predictable (Cmin, AUC) and comparable (t1/2, CLss) between both regimens (see table below). Preliminary skin PD studies show no major difference in the EGFR-signaling inhibition by C in the q1w and q2w regimens. Conclusions: C can be safely administered in a q2w regimen at 400 and 500 mg/m2. The MTD of q2w C has not yet been reached and dose escalation is ongoing. Our findings show no major difference in PK and PD profiles with the q1w and q2w schedules. C given q2w may be an alternative and convenient schedule of administration. [Table: see text] [Table: see text]

3007 Background: Everolimus (E), an oral derivative of rapamycin, inhibits mTOR, a protein kinase downstream of PI3K and Akt, involved in the regulation of cell growth, proliferation and survival. ...

3112 Background- Everolimus inhibits mTOR and has anti-tumor activity in vitro and in vivo. Interestingly, E induces up-stream phosphorylated Akt (p-Akt), which could in turn mediate resistance to ...

3077 Background: Matuzumab is a humanized monoclonal antibody binding to the EGFR with higher affinity than natural ligands (eg. TGF alpha,EGF) leading to inhibition of signal transduction pathways...

3028 Background: AEE788 is an orally active, small-molecule, multitargeted kinase inhibitor with potent inhibitory activity against multiple tyrosine kinases, including EGFR, HER2, and VEGFR2. This...

3543 Background: In preclinical models, EKB-569 has shown growth inhibition of EGFR-expressing colorectal tumor cell lines with at least additive inhibition when combined with chemotherapy. Methods...

3074 Background: mTOR plays a critical role in transducing proliferative signals mediated through the PI3-K/AKT pathway. We have previously demonstrated that everolimus (E) decreased p-4E-BP1 and p-p70S6K levels, the two known downstream targets of mTOR, and inhibited tumor cell proliferation. Interestingly, E-treatment increased the p-AKT levels. We investigated whether the blockade of EGFR by gefitinib (G) could prevent E-induced p-AKT and to determine the optimal schedule of combining E and G. Methods: Cell growth inhibition and western blot assays were performed on BT-474, MDA-MB-468 and DU-145 cancer cell lines. For the simultaneous exposure, cells were incubated for 72 hours with G and/or E, at doses including the IC50 for each drug and cell type. For the sequential exposure, cells were treated with G for 48 hours, washed twice and incubated for a further 48 hours with E, and viceversa. Results were analysed using the combination index (CI) method. Results: The simultaneous treatment of BT-474 and D...

9524 Background: Activation of the PI3-K/Akt/mTOR signal transduction pathway contributes to the development and progression of tumors by prevention of apoptosis and deregulation of the cell cycle. Dissecting the molecular events of this pathway may provide instrumental knowledge with implications for the development of PI3K/Akt/mTOR inhibitors and multi-signalling inhibitory strategies. Methods: We have analysed 121 paraffin-embedded human malignant tumors: 25 gastric, 25 colon, 23 breast, 19 prostate, 19 glioblastoma and 10 pancreas, with a complete immunohistochemistry profile including multiple phosphorylated (p) downstream proteins: pAkt, p4EBP1, pp70S6K and pS6K. The levels of expression were evaluated as percentage and intensity of stained cells (Hscore). Results: Globally, there was a significant correlation between the levels of pAkt, p4EBP1, pp70S6K and pS6K (Spearman's test, p

3061 Background: Monoclonal antibody EMD 72000 blocks ligand binding and inhibits EGFR activation and signal transduction. EGFR is overexpressed in up to 90% of pancreatic cancers. We studied safet...

Background DEK is a transcription factor involved in stabilization of heterochromatin and cruciform structures. It plays an important role in development and progression of different types of cancer. This study aims to analyze the role of DEK in metastatic colorectal cancer. Methods Baseline DEK expression was firstly quantified in 9 colorectal cell lines and normal mucosa by WB. SiRNA-mediated DEK inhibition was carried out for transient DEK silencing in DLD1 and SW620 to dissect its role in colorectal cancer aggressiveness. Irinotecan response assays were performed with SN38 over 24 hours and apoptosis was quantified by flow cytometry. Ex-vivo assay was carried out with 3 fresh tumour tissues taken from surgical resection and treated with SN38 for 24 hours. DEK expression was determined by immunohistochemistry in 67 formalin-fixed paraffin-embedded tumour samples from metastatic colorectal cancer patients treated with irinotecan-based therapy as first-line treatment. Results The DEK oncogene is overexpressed in all colorectal cancer cell lines. Knock-down of DEK on DLD1 and SW620 cell lines decreased cell migration and increased irinotecan-induced apoptosis. In addition, low DEK expression level predicted irinotecan-based chemotherapy response in metastatic colorectal cancer patients with KRAS wild-type. Conclusions These data suggest DEK overexpression as a crucial event for the emergence of an aggressive phenotype in colorectal cancer and its potential role as biomarker for irinotecan response in those patients with KRAS wild-type status.

The mechanisms that allow breast cancer (BCa) cells to metabolically sustain rapid growth are poorly understood. Here we report that BCa cells are dependent on a mechanism to supply precursors for intracellular lipid production derived from extracellular sources and that the endothelial lipase (LIPG) fulfils this function. LIPG expression allows the import of lipid precursors, thereby contributing to BCa proliferation. LIPG stands out as an essential component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. LIPG is ubiquitously and highly expressed under the control of FoxA1 or FoxA2 in all BCa subtypes. The downregulation of either LIPG or FoxA in transformed cells results in decreased proliferation and impaired synthesis of intracellular lipids., Deregulation of lipid metabolism in cancer cells is critical to the maintenance of certain malignant features. Here, the authors show that the proliferation of breast cancer cells depends upon the extracellular activity of the endothelial lipase enzyme LIPG whose expression is regulated by the FoxA family of transcription factors.

// Aranzazu Fernandez-Martinez 1, 2 , Tomas Pascual 1, 2 , Giuseppe Perrone 3 , Serafin Morales 4 , Juan de la Haba 5 , Milagros Gonzalez-Rivera 6 , Patricia Galvan 1, 2, 7 , Francesca Zalfa 3 , Michela Amato 3 , Lucia Gonzalez 8 , Miquel Prats 9 , Federico Rojo 10 , Luis Manso 11 , Laia Pare 1, 2 , Immaculada Alonso 1 , Joan Albanell 12 , Ana Vivancos 7 , Antonio Gonzalez 13 , Judit Matito 7 , Sonia Gonzalez 14 , Pedro Fernandez 1 , Barbara Adamo 1, 2 , Montserrat Munoz 1, 2 , Margarita Viladot 1, 2 , Carme Font 1, 2 , Francisco Aya 1, 2 , Maria Vidal 1, 2 , Rosalia Caballero 15 , Eva Carrasco 15 , Vittorio Altomare 3 , Giuseppe Tonini 3 , Aleix Prat 1, 2, 7 , Miguel Martin 6 1 Medical Oncology Department, Hospital Clinic of Barcelona, Barcelona, Spain 2 Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research Institute, Barcelona, Spain 3 Department of Medicine, Universita Campus Bio-Medico di Roma, Rome, Italy 4 Medical Oncology Deparment, Arnau de Vilanova de Lleida Universitary Hospital, Lleida, Spain 5 Medical Oncology Department, Reina Sofia University Hospital, Cordoba, Spain 6 Medical Oncology Department, Instituto de Investigacion Sanitaria Gregorio Maranon (IISGM), Universidad Complutense, Madrid, Spain 7 Vall d'Hebron Institute of Oncology, Barcelona, Spain 8 Medical Oncology Department, Quiron Hospital, Madrid, Spain 9 Master of Breast Pathology, University of Barcelona, Barcelona, Spain 10 Pathology Department, Fundacion Jimenez Diaz Health Research Institute (IIS-FJD), Madrid, Spain 11 Medical Oncology Department, Doce de Octubre Hospital, Madrid, Spain 12 Medical Oncology Department, Hospital del Mar, Barcelona, Spain 13 Medical Oncology Department, MD Anderson Cancer Center, Madrid, Spain 14 Medical Oncology Department, Mutua de Terrassa Hospital, Barcelona, Spain 15 Spanish Breast Cancer Research Group Grupo Espanol de Investigacion en Cancer de Mama (GEICAM), Madrid, Spain Correspondence to: Miguel Martin, email: mmartin@geicam.org Aleix Prat, email: aprat@vhio.net Keywords: PAM50/Prosigna, breast cancer, Ki67, estrogen receptor-positive/HER2-negative Received: October 19, 2016 Accepted: January 27, 2017 Published: February 27, 2017 ABSTRACT PAM50/Prosigna gene expression-based assay identifies three categorical risk of relapse groups (ROR-low, ROR-intermediate and ROR-high) in post-menopausal patients with estrogen receptor estrogen receptor-positive (ER+)/ HER2-negative (HER2-) early breast cancer. Low risk patients might not need adjuvant chemotherapy since their risk of distant relapse at 10-years is below 10% with endocrine therapy only. In this study, 517 consecutive patients with ER+/HER2- and node-negative disease were evaluated for Ki67 and Prosigna. Most of Luminal A tumors (65.6%) and ROR-low tumors (70.9%) had low Ki67 values (0-10%); however, the percentage of patients with ROR-medium or ROR-high disease within the Ki67 0-10% group was 42.7% (with tumor sizes ≤2 cm) and 33.9% (with tumor sizes > 2 cm). Finally, we found that the optimal Ki67 cutoff for identifying Luminal A or ROR-low tumors was 14%. Ki67 as a surrogate biomarker in identifying Prosigna low-risk outcome patients or Luminal A disease in the clinical setting is unreliable. In the absence of a well-validated prognostic gene expression-based assay, the optimal Ki67 cutoff for identifying low-risk outcome patients or Luminal A disease remains at 14%.