Amphiregulin Levels Research Articles

Stress-induced neuroprotective effects of epiregulin and amphiregulin.

Members of the epidermal growth factor family play important roles in the regulation of cell growth, proliferation, and survival. However, the specific roles of each epidermal growth factor family member with respect to brain injury are not well understood. Gene chip assay screens have revealed drastic increases in the expression of the epidermal growth factor family members amphiregulin and epiregulin following lipopolysaccharide stimulation, which activates an immune response. Both immune activity and endoplasmic reticulum stress are activated during cerebral ischemia. We found that the expression levels of amphiregulin and epiregulin were significantly increased under conditions of cerebral ischemia. Because endoplasmic reticulum stress increased the expression of amphiregulin and epiregulin in glial cells, endoplasmic reticulum stress may be a key mediatory factor of pathophysiological activity. Recombinant epiregulin and amphiregulin proteins effectively inhibited endoplasmic reticulum stress and the subsequent induction of neuronal cell death. Therefore, the upregulation of the epidermal growth factor family members epiregulin and amphiregulin may play a critical role in preventing endoplasmic reticulum stress-induced cell death, thus providing a potential therapy for brain injury.

EGFR ligands and ERCC1 mRNA expression to predict clinical outcome in Japanese (JPN) patients (pts) with metastatic colorectal cancer (mCRC) harboring overexpressed EGFR and KRAS exon 2 wild-type (KRAS wt) treated with cetuximab (cet) plus oxaliplatin-based chemotherapy (JACCRO CC-05/06 AR).

618 Background: Previous studies have reported that EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), are potential predictive markers for cet and high ERCC1 expression is associated with resistance to platinum-containing chemotherapy. However, there are still few studies to assess predictive values of these expression levels in JPN mCRC pts. The aim of this study was to evaluate the values in JPN mCRC pts treated with cet. Methods: This study enrolled 77 pts with tissue available from 2 prospective clinical trials evaluating combination of cet with oxaliplatin-based chemotherapy as first-line treatment in pts with KRAS wt and EGFR-expressing tumors, modified FOLFOX6 (n=28/57, UMIN000004197) and SOX (n=49/67, UMIN000007022). Total RNA isolated by macro-dissection from tissue was screened by RT-PCR for mRNA expression levels of AREG, EREG, and ERCC1, the cut-off values of which were based on the optimal cut-off value using the maximal chi-square approach on tumor response with adjusted p values. The same cut-off values were used to evaluate associations with progression-free survival (PFS) and overall survival (OS). Results: Median age and follow-up time were 63 (range: 39-79) years old and 24.7 (range: 5.5-39.3) months, respectively. In 73 evaluable pts, response rate (RR) was 77 % (95% CI, 67 to 86), but no significant association was seen in RR. Pts with high AREG expression (>1.59) had significantly longer PFS (11.6 vs. 6.6 months, HR: 0.53, logrank p=0.047) but not OS in 67 assessable pts. Pts with low ERCC1 expression (≤1.35) had significantly longer OS (42.8 vs. 22.0 months, HR: 2.46, logrank p=0.014) but a trend toward longer PFS (11.6 vs. 8.9 months, HR: 1.63, logrank p=0.092) in 68 assessable pts. On the other hand, high EREG expression (>3.21) was not associated with clinical outcome in 64 assessable pts. Conclusions: Our study identified predictive roles for AREG and ERCC1 mRNA expression in JPN mCRC pts treated with cet plus oxaliplatin. However, extended RAS mutations analysis is warranted (UMIN000010635).

Amphiregulin (AREG) expression and response to first-line panitumumab (pmab) plus FOLFIRI in metastatic colorectal cancer (mCRC).

731 Background: Biomarker analyses have shown that patients (pts) with RAS wild-type (WT) mCRC can achieve overall survival (OS) benefits with first-line pmab plus chemotherapy. Other biomarkers may exist that could optimize pt selection. Epidermal growth factor receptor ligand (eg AREG) levels have been correlated with OS during anti-EGFR therapy. Here we investigate the relationship between AREG expression and treatment outcome in a single-arm first-line mCRC study of pmab + FOLFIRI. Methods: Qualified reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were used to measure AREG RNA expression in archival formalin-fixed, paraffin embedded tumor samples from mCRC pts in two pmab trials (STEPP and 314). The STEPP analysis was used to establish a cut-off point in AREG expression that identified the best responders. This cut-off was applied prospectively to samples previously analyzed for KRAS in the 314 trial. Using the KRASMT subgroup as a non-responding comparator, Cox proportional hazards (PH) models were used to evaluate AREG expression levels as a continuous covariate. Decision curves were used to estimate the progression-free survival (PFS) hazard ratio (HR) with increasing levels of baseline AREG expression. Results: In the 314 trial 100 pts had evaluable AREG levels. Among 50 KRAS WT pts, high AREG expression was associated with objective response (OR) (Table). The high AREG group had better PFS HRs (KRAS WT/KRAS MT: 0.30 [95% CI, 0.12–0.75]) compared with the low AREG group (PFS HR 0.49 [95% CI 0.21–1.1]). There was a significant biomarker-by-AREG expression interaction in the Cox PH model (p=0.03). Conclusions: Treatment decision curves based on the PH model suggest that most KRAS WT patients express AREG at levels where treatment benefit is predicted. Future analysis of samples from a RASWT population may provide further insights. Clinical trial information: NCT00508404. [Table: see text]

Abstract 557: Noninvasive analysis of acquired resistance to EGFR-TKI

Abstract BACKGROUND Epidermal growth factor receptor (EGFR) T790M mutation is associated with EGFR tyrosine kinase inhibitors (EGFR-TKIs) resistance in non-small cell lung cancer (NSCLC). However, the tissue availability limits the genotyping of EGFR T790M mutation in the clinical setting. The aims of this study are to develop a blood-based, non-invasive approach to detect the EGFRT790M mutation in advanced NSCLC patients. Additionally, plasma concentrations of HGF, MET and amphiregulin which are also resistant factors of EGFR-TKIs EXPERIMENTAL DESIGN Plasma sample were collected from NSCLC patients with activating EGFR mutations, who had resistant to EGFR-TKIs treatment. EGFR T790M mutations were detected using droplet-digital PCR (ddPCR) system from plasma DNA at resistance to EGFR-TKI. Plasma concentrations of HGF, soluble MET (sMET) and amphiregulin were determined using enzyme-linked immunosorbent assay. The relationships between the EGFR T790M mutation status and the plasma concentrations, progression-free survival (PFS) overall survival (OS) were analyzed. RESULTS Twenty-one (35%) of the patients had EGFR T790M mutation in plasma DNA using ddPCR system. The levels amphiregulin tended to be increased at the resistance in the patients with EGFR T790M mutation. The PFS and OS of the patients with EGFR T790M mutation was significantly shorter than that of the patients without EGFR T790M mutation (P =0.03 and 0.04). The PFS of the patients who had high amphiregulin at the resistance and the OS of the patients who had high amphiregulin at pre-treatment were shorter than these of t low amphiregulin (P = 0.05 and 0.05) CONCLUSIONS The ddPCR system is a detectable method for determining the plasma EGFR T790M mutation status. The combination of EGFR T790M mutation status by ddPCR and the amphiregulin levels may aid in monitoring drug resistance and in predicting a prognosis. Citation Format: Hideharu Kimura, Makoto Nishio, Tsutomu Daito, Kazuto Nishio. Noninvasive analysis of acquired resistance to EGFR-TKI. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 557. doi:10.1158/1538-7445.AM2014-557

Abstract 1842: Synergistic inhibition of ALK inhibitor-resistant lung cancer cells by dual-targeting EGFR and ALK pathways

Abstract Background: Despite the excellent initial responses of ALK-rearranged lung cancer to the ALK tyrosine kinase inhibitors (TKIs), most patients develop acquired resistance through several mechanisms, including ALK amplification, mutation or activation of alternative signaling pathways. In this study, we investigated the effect of dual-targeting EGFR and ALK pathways in NSCLC cells that are resistant to ALK inhibitors. Methods: The cell proliferation, apoptosis and cell cycle analysis were measured by spectrophotometry and flow cytometry. The combination effect of EGFR TKI (afatinib or dacomitinib) and ALK TKI (crizotinib or LDK378) was evaluated using a combination index (CI). We analyzed EGFR ligand levels by ELISA and expression of ALK, EGFR and its downstream molecules were detected by Western blot assay after exposure to TKIs. Results: NCI-H3122CR1 that was resistant to crizotinib and NCI-H3122 cell lines became resistant to LDK378 through the step-wise increase of the drug, resulting in H3122CR1LR1 and H3122LR1 cells, respectively. The resistant cell lines exhibited 10-14-fold higher IC50 values for crizotinib or LDK378 compared with parental cells. The genetic alterations of ALK or EGFR genes were not found in H3122CR1, H3122LR1, and H3122CR1LR1 cells. We analyzed EGFR signaling pathways in these cells to determine their roles in mediating ALK inhibitor resistance. We observed that H3122CR1, H3122LR1 and H3122CR1LR1 cells had higher phospho-EGFR than parental cells. In these cells, mRNA and protein levels of amphiregulin and EGF were up-regulated. Combined treatment with EGFR TKI and LDK378 synergistically inhibited cell growth (CI<1) and induced apoptosis in H3122LR1 and H3122CR1LR1 cells. Similar results were observed in H3122CR1 and SNU-2535 harboring ALK G1269A mutation cells, when exposed to afatinib and crizotinib. The combination of EGFR and ALK TKIs decreased the phosphorylation of several downstream molecules in H3122LR1 and H3122CR1LR1 cells, whereas EGFR or ALK TKI alone did not reduce the phosphorylation levels. Conclusion: Our results suggest that dual inhibition of ALK and EGFR effectively abrogate ALK and its downstream signals and induce apoptosis in ALK TKIs-resistant, ALK-rearranged NSCLC cells. Therefore, dual-targeting of EGFR and ALK might be a promising strategy for ALK-rearranged NSCLC patients who showed acquired resistance to crizotinib and/or LDK378. Citation Format: Soyeon Kim, Tae Min Kim, Yong-Oon Ahn, Bhumsuk Keam, Se-Hoon Lee, Dae Seog Heo, Dong-Wan Kim. Synergistic inhibition of ALK inhibitor-resistant lung cancer cells by dual-targeting EGFR and ALK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1842. doi:10.1158/1538-7445.AM2014-1842

Abstract 3297: PTEN loss enhances amphiregulin-specific signaling and gene expression in triple-negative breast cancer

Abstract The epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer, and approximately half of all basal breast cancers show autocrine activation of EGFR by amphiregulin (AREG). When AREG is the activating ligand, the dynamics of EGFR signaling are different than those of EGF-bound EGFR. AREG-ligated EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To further explore the role of amphiregulin in cell signaling, we performed reverse-phase protein array (RPPA) analysis on SUM149 cells, a highly tumorigenic and metastatic cell line that expresses high levels of AREG and EGFR, and MCF10A cells, a non-transformed mammary epithelial cell line cultured in the presence of either EGF or AREG. This analysis identified a number of signaling components mediated specifically by amphiregulin, including decreased caspase expression, increased Gab2 expression, elevated fibronectin levels and marked changes in integrin expression. In addition, AREG-mediated EGFR signaling resulted in increased expression and activation of c-src. Thus, a major effect of AREG signaling in both cell lines was enhanced activation of the canonical focal adhesion signaling pathway. In addition to increased expression of EGFR and AREG, SUM149 cells are known to have a genomic alteration at the PTEN locus resulting in complete abrogation of PTEN message and protein expression. RPPA analysis of signaling in SUM-149 cells confirmed this PTEN loss and, not surprisingly, revealed elevated AKT activity. In addition, several proteins in the WNT signaling pathway were altered in SUM-149 cells, including reduced expression of GSK3β, increased phosphorylation of GSK3β, and increased expression of Dvl. These results suggest enhanced Wnt-beta catenin activity in SUM-149 cells that is likely the result of enhanced AKT activity resulting from PTEN loss. Although we previously had difficulty measuring Wnt signaling in SUM-149 cells in monolayer culture, mammosphere assays performed with SUM149 cells expressing a Wnt reporter construct confirmed that Wnt signaling was highly active in mammospheres in 3D culture. We also analyzed the genes that are regulated in expression by EGFR in MCF-10A cells in the presence of EGF or AREG, and in SUM-149 cells. These results identified AREG-specific gene expression profiles in the isogenic system. In addition, SUM-149 cell-specific genes regulated by EGFR were also identified, some of which are likely to be the result of PTEN loss and enhanced AKT signaling in these cells. Finally, we found that AREG knockdown in SUM-149 cells significantly reduced the tumorigenicity of SUM149 cells in NOD-SCID mice. Analysis of signaling and gene expression in these AREG knock-down cells are consistent with the importance of AREG expression levels and of the modulation of the focal adhesion canonical pathway in tumor potential of the cells in vivo. Citation Format: Christiana Kappler, Robert Wilson, Bridget Varughese, Stephen P. Ethier. PTEN loss enhances amphiregulin-specific signaling and gene expression in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3297. doi:10.1158/1538-7445.AM2014-3297

EGFR gene gain and PTEN protein expression are favorable prognostic factors in patients with KRAS wild-type metastatic colorectal cancer treated with cetuximab

Cetuximab is a monoclonal epidermal growth factor receptor (EGFR)-targeting antibody, used in the treatment of colon cancer. KRAS mutation status is strongly predictive of cetuximab efficacy, but more predictive factors are needed for better patient selection. PTEN is a downstream inhibitor of the EGFR pathway and has been evaluated as a predictive factor of cetuximab efficacy in colorectal cancer. Formalin-fixed paraffin-embedded tumor tissue samples were collected from 226 patients with advanced or metastatic colorectal cancer that had been treated with cetuximab. Clinical information was collected retrospectively from the patients' medical records. After central evaluation, 147 cases with adequate material were eligible for further evaluation. EGFR and PTEN status was evaluated with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Data were associated with cetuximab treatment outcome. Additional analysis was performed with previously published data on PIK3CA, BRAF and KRAS mutation status and EGFR ligand amphiregulin (AREG) and epiregulin intratumoral mRNA expression levels. PIK3CA mutation status and PTEN protein expression were also analyzed as a single complex parameter, to evaluate the predictive value of PI3K/PTEN axis dysfunction as one entity. Analysis showed a borderline association of overall response rate (ORR) and time to progression (TTP) with EGFR protein overexpression by IHC (p = 0.059 and p = 0.057, respectively) and a positive association of EGFR gain by FISH (found in only five cases) with longer TTP (p = 0.026). No association was found between ORR or TTP and PTEN IHC or FISH status. Comparative analysis with previously published data showed that PTEN protein expression is associated with longer TTP in patients with wild-type (WT) KRAS (p = 0.036) and especially in the ones with elevated AREG levels (p = 0.046), as well as in patients with both KRAS and BRAF WT (p = 0.019). Patients with both PIK3CA WT and PTEN protein expression had significantly longer TTP (p = 0.010) versus all others, in the absence of BRAF and KRAS mutations, a finding which persisted in the KRAS WT/AREG high subgroup (p = 0.046). In this cetuximab-treated colorectal cancer population, EGFR gain was associated with better outcome and PTEN protein expression with longer TTP in KRAS WT, KRAS WT/AREG high and KRAS/BRAF WT subpopulations. Cetuximab efficacy is greater with intact and activated EGFR signaling, without activating mutations of KRAS/BRAF and in the presence of preserved PTEN inhibitory activity upon the PI3K/AKT pathway. These results reflect a solid biological rationale and warrant further evaluation of the predictive role of PTEN in prospective studies.

IL-9–mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation

IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)-deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation.

Intermittent versus continuous erlotinib with concomitant modified “XELOX” (q3W) in first‐line treatment of metastatic colorectal cancer

This study evaluated the activity of 2 schedules of erlotinib in combination with chemotherapy, and the prognostic significance of serum amphiregulin (AREG) and transforming growth factor alpha (TGFa) in metastatic colorectal cancer. A total of 60 untreated patients were randomized to a "continuous" (CON; erlotinib 100 mg daily) or an "intermittent" (INT; erlotinib 150 mg on alternate day on day 2 to 14, then 150 mg daily on days 15 to 21) schedule of erlotinib with a modified XELOX (capecitabine plus oxaliplatin) regimen. Serum levels of AREG and TGFa were determined serially. Baseline characteristics were similar between the 2 arms. Of the 58 patients evaluated for response, there was a nonsignificant trend toward a slightly higher overall response rate in the INT arm (66.7%) versus the CON arm (56.7%). At a median follow-up of 2.8 years, the median overall survival was 18.8 months (95% confidence interval = 11.3-22.9 months) and 20.7 months (95% confidence interval = 12.5-31 months, P = .19) for the CON and INT arm, respectively. KRAS mutation did not predict drug response. The 2 arms did not differ significantly in toxicity. Baseline serum TGFa was an independent predictor of progression-free survival, whereas a drop in serum TGFa and AREG levels following 3 to 4 cycles of treatment were associated with shorter progression-free survival and overall survival, respectively. The intermittent erlotinib schedule was associated with a higher response rate, although this is not statistically significant. Serum TGFa and AREG levels have prognostic significance in erlotinib-treated patients with colorectal cancer, and further studies are warranted.

Evaluation of serum Amphiregulin levels in breast cancer patients and cancer-free controls

BackgroundExpression of the Epidermal Growth Factor Receptor ligand, Amphiregulin, has been associated with estrogen receptor positive breast cancer. As Amphiregulin is proteolytically released from the surface of breast cancer cells, we investigated the levels of Amphiregulin in the serum of breast cancer patients and cancer-free women to evaluate its potential utility as a breast cancer biomarker.FindingsSerum Amphiregulin levels were quantified by ELISA from 125 cancer-free women and 114 breast cancer patients. No significant association between serum Amphiregulin levels and breast cancer status was detected at two cut-points evaluated.ConclusionsMeasurement of serum Amphiregulin levels lacks the necessary sensitivity and specificity for breast cancer screening in the general population.

Prospective analysis of the early modulation of plasma amphiregulin (AR) during treatment with cetuximab and irinotecan in irinotecan-refractory metastatic colorectal cancer (mCRC) patients (pts).

3602 Background: The potential role of AR tissue levels in the prediction of benefit from anti-EGFRs in mCRC pts was suggested by retrospective series. Preclinical and preliminary clinical experiences showed a modulation of plasma EGFR ligands during the treatment with cetuximab. Previous data by our group evidenced that a significant increase of plasma AR occurred one hour after the administration of cetuximab and higher increases were associated with worse clinical outcome in KRAS and BRAFwt irinotecan-refractory mCRC pts receiving cetuximab and irinotecan. Methods: We designed a prospective confirmatory study in the same setting of mCRC pts. To detect a HR for PFS of 2.3 for pts with high AR levels one hour after the administration of cetuximab (1hr-AR) compared to those with low levels, with two-sided a=0.05 and b=0.2, 45 events were required. The median value was adopted as cut-off. Plasma AR levels were assessed by means of validated ELISA kits. Results: Forty-nine KRAS and BRAFwt pts were included. A significant early increase of AR levels was observed (median increase +24.7%; median levels of baseline AR and 1hr-AR: 18.06 and 24.06 pg/mL, respectively; Wilcoxon signed rank test, p<0.0001). At a median follow-up of 20.4 mos, median PFS and OS were 4.6 and 12.1 mos, respectively. No differences in PFS or OS were observed according to 1hr-AR levels (median PFS 5.5 vs 4.6 mos, HR: 0.76 [95%CI:0.40-1.32], p=0.322; median OS: 15.6 vs 13.4 mos, HR:0.77 [95%CI:0.36-1.62], p=0.485). Conclusions: This prospective experience confirms that AR early increases one hour after the administration of cetuximab. Underlying biological mechanisms should be investigated. Nevertheless, this modulation of AR does not predict clinical outcome. Our work underlines the need to prospectively validate retrospective findings in independent series, to assess their reliability. Clinical trial information: 2008-003160-19.

Association of a polymorphism in amphiregulin with worse prognosis in metastatic colorectal cancer patients treated with cetuximab.

e14543 Background: Accumulating data suggests that Amphiregulin (AREG) and Epiregulin (EREG), ligands of the EGFR pathway, are potential prognostic and predictive markers in metastatic colorectal cancer (mCRC). Elevated AREG and EREG gene expression levels are predictive of improved response rates in mCRC patients treated with cetuximab and longer overall survival (OS) in patients with mCRC who did not receive cetuximab. Since polymorphisms in AREG and EREG may affect their expression or function, we tested whether AREG and EREG polymorphisms were associated with clinical outcome in mCRC patients treated with cetuximab. Methods: Seventy-three patients (n=73) with histopathologically-confirmed mCRC with Kras/Braf wild type were enrolled in Italy. These patients were treated with cetuximab monotherapy or irinotecan plus cetuximab as second or third line treatment. Genomic DNA was extracted from blood and 4 SNPs in AREG and 5 SNPs in EREG were analyzed by direct DNA-sequencing. Results: The median follow-up period was 13.3 months in this cohort. The median progression-free survival (PFS) and OS were 5.2 and 12.9 months respectively. The results indicate that the intronic AREG polymorphism rs28364983 A/C was associated with shorter PFS and OS. Patients with homozygous C/C (n=2) showed a median PFS of 2.3 months vs. 5.3 months for patients harboring at least one-A allele (n=71) (HR: 4.26 [95%CI: 0.98-18.5], p=0.05). Patients with homozygous C/C showed a median OS of 3.0 months vs. 13.1 months for patients harboring at least one-A allele (HR: 3.98 [95%CI: 0.93-16.9], p=0.062). An independent cohort of thirty-three patients was used to validate these initial findings and showed that patients with homozygous C/C (n=3) had a median PFS of 2.3 months vs. 3.8 months for patients harboring at least one-A allele (n=103) (HR: 3.79 [95%CI: 1.16-12.3], p=0.027) and a median OS of 3.0 months vs. 8.4 months (HR: 3.01 [95%CI: 0.93-9.71], p=0.065), respectively. Conclusions: AREG rs28364983 predicted worse outcome in mCRC patients treated with cetuximab; however, further analyses are necessary to understand the mechanism and further validate the prognostic and/or predictive role of this polymorphism.

Association of AREG exon 1 polymorphism with worse prognosis in Japanese and U.S. patients with gastric cancer.

e15022 Background: Recent data suggest that Amphiregulin (AREG) and Epiregulin (EREG), ligand of EGFR pathway, is possibly a prognostic and predictive marker in gastrointestinal cancer. Previous data have shown that higher AREG and EREG gene expression levels are associated with better survival in metastatic colorectal cancer as well as gastric cancer (GC). Since polymorphisms in AREG and EREG may affect their gene expression levels or change their functions, we tested the hypothesis whether these polymorphisms could predict outcome in two ethnically and epidemiologically different GC cohorts from Japan and the US. Methods: One-hundred and sixty-nine Japanese (n=169), as a training set, and one-hundred and thirty-seven (n=137) the US patients, as a validation set, with histopathologically-confirmed localized (stage Ib to IV; AJCC-6th) GC were enrolled between 2002 and 2010. Four functional SNPs in AREG and five SNPs in EREG were selected. Genomic DNA was extracted from blood or tissue, and all samples were analyzed by PCR-based direct DNA-sequencing. Results: The median follow-up periods were 4.0 years in the Japanese cohort and 3.3 years in the US cohort, respectively. The median DFS and OS in the Japanese cohort were 4.8 and 5.8 years, whereas the median TTR and OS in the US cohort were 2.8 and 4.7 years, respectively. Only exon 1 polymorphism rs1615111 G/A was associated with outcome in Japanese cohort. Patients heterozygous G/A (n=23) showed a median OS of 2.7 years vs. 20.1 years for patients homozygous G/G (n=146) (HR: 1.83 [95%CI: 1.02-3.30], p=0.039). This result was successfully validated in the US cohort. Patients harboring at least one-A allele (n=14) showed a median OS of 2.8 years vs. 5.4 years for patients homozygous G/G (n=111) (HR: 2.54 [95%CI: 1.09-5.89], p=0.018). In the pooled multivariate analysis, these results were still conserved (HR=2.24 [95%CI: 1.36- 3.67], p=0.0015). Conclusions: AREG exon 1 polymorphism rs161511 may be negative prognostic factor in GC regardless ethnicity and regional differences in gastric cancer. Further clinical validation of SNPs in EGFR ligands are warranted.

Abstract 530: Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells.

Abstract Approximately half of all basal type breast cancers show evidence of autocrine activation of the epidermal growth factor receptor (EGFR) by amphiregulin (AREG). In addition, AREG expression is associated with aggressive and chemoresistant forms of breast cancer. Studies in our laboratory show that when AREG is the activating ligand, EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To investigate the effect of AREG-mediated activation of EGFR on the phenotype of AREG-expressing cancers, AREG expression was knocked-down in SUM149 cells, a highly tumorigenic and metastatic cell line isolated from a basal subtype inflammatory breast cancer, which expresses high levels of AREG and overexpresses EGFR. Microarray analysis of AREG knockdown cells revealed a number of relevant pathways affected by AREG-EGFR signaling. These pathways include Notch signaling, MAPK activation, IL-1 signaling and multiple components of the Wnt signaling pathway. In particular, knockdown of AREG or inhibition of EGFR signaling in AREG stimulated cells resulted in the upregulation of DKK-1, an antagonist of canonical Wnt signaling, and the expression of several WNT target genes. Flow cytometric analysis of these cells demonstrated that AREG knockdown resulted in a significant reduction in CD44+ cells while simultaneously increasing the non-stem cell fraction of CD44-/CD24+ cells. Because CD44hi/CD24lo is characteristic of undifferentiated cells that have the potential to initiate tumor formation, the tumorigenicity of these cells was investigated by transplanting parental and AREG knock-down SUM149 cells into the mammary fat pads of NOD-SCID mice. The tumorigenic activity of the AREG knock-down derivatives was significantly reduced compared to the parental SUM149 cells. In particular, the AREG knockdown clone that expressed the lowest level of AREG expression and the highest level of DKK-1 expression failed to form tumors in NOD-SCID mice. To investigate the link between AREG-mediated activation of EGFR, receptor trafficking, and downstream signaling pathways that are responsible for changes in expression of WNT pathway genes, we are investigating differences in EGFR phosphorylation and downstream signaling stimulated by AREG versus EGF. In conjunction with our previous findings that AREG-mediated EGFR signaling results in activation of NF-κβ and the upregulation of IL1α/β, our current results suggest a link between these pathways, WNT signaling, and tumorigenicity in SUM-149 cells. Citation Format: Christiana S. Kappler, Stephen P. Ethier. Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2013-530

Abstract 2401: Evaluation of circulating serum Amphiregulin levels in patients with breast cancer.

Abstract Amphiregulin (AREG), a ligand for Epidermal Growth Factor Receptor (EGFR), has been implicated as an effector of ERα signaling in mammary gland development and as a candidate ERα effector in human breast cancers. Analysis of 13 luminal subtype breast cancer cell lines and human breast tumors showed a strong correlation between ERα status and high AREG levels. A pilot study using healthy and breast cancer women's serum showed that circulating AREG levels were higher in the ERα positive patients as compared to healthy individuals. Therefore we hypothesized that Amphiregulin is a potential biomarker and/or therapeutic target for ERα positive breast cancers. To test our hypothesis, we are currently evaluating AREG as a potential serum biomarker of breast cancer progression and as an indicator of response to treatment by analyzing a cohort of breast cancer patients (n=225) stratified by stage of disease and subtypes (hormone receptor status). In this study, we want to determine if tumor-derived AREG is released into the systemic circulation and whether it may be a useful serum biomarker for the presence of breast cancer and for response to therapy. In the first phase, serum Amphiregulin was measured by an ELISA assay to establish the reference range in 125 healthy females. Fifty-five percent of the healthy women had no detectable circulating AREG (n=69), and only ten women had levels exceeding 500 pg/mL. Serum AREG levels did not vary significantly during the menstrual cycle. We determined that the 95th and 90th percentile was >1575 pg/ml and >471 pg/ml, respectively, to use as a comparison against the AREG levels of the breast cancer patients. We are analyzing serum AREG levels in breast cancer patient serum samples of three different cohorts: women with no evidence of disease after more than 1 year post-surgery (Cohort 1), women with locally advanced or stage IV with disease progression (Cohort 2) and women with operable disease that will have surgery and subsequent chemotherapy and/or endocrine therapy (Cohort 3). Each cohort was further stratified by hormone receptor status. In addition to determining the baseline AREG levels, for Cohort 3, AREG levels will be measured post-surgery and at different timepoints during treatment. Interim analysis of the different cohorts showed a significance difference (p<0.05) between healthy individuals and patients with ERα+ breast tumors when comparing detectable versus undetectable AREG levels (threshold of detection: 20 pg/ml). Interestingly, longitudinal sampling of one patient with very high levels (18,300 pg/ml at diagnosis) showed a 37% decline following tumor resection and a 60% decline after 9 months of endocrine therapy. In summary, determining the potential role of AREG as predictive biomarker for therapy and/or as therapeutic target by the completion of this clinical study, will contribute to the quest for better predictive biomarkers in breast cancer and better management of the disease. Citation Format: Esther A. Peterson, Paraic A. Kenny. Evaluation of circulating serum Amphiregulin levels in patients with breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2013-2401

Abstract 2386: Amphiregulin confers trastuzumab resistance by activating PI3K/Akt pathway in HER2-positive breast cancer.

Abstract Background: Amphiregulin is a ligand for the epidermal growth factor receptor (EGFR). Human epidermal growth factor receptor 2 (HER2) shares common signal pathways and forms a heterodimer with EGFR. In this study, we investigated the effect of amphiregulin on trastuzumab therapy in HER2-positive breast cancer. Methods: We analyzed serum amphiregulin levels by enzyme-linked immunosorbent assay (ELISA) from baseline serum samples obtained from HER2-positive metastatic breast cancer patients who received first-line trastuzumab plus taxane chemotherapy. In addition, in vitro experiments were performed to elucidate the biologic mechanism of clinical findings related to amphiregulin using SK-BR-3 and BT-474 cell lines. Results: Between October 2004 and July 2009, a total of 50 women with HER2-positive metastatic breast cancer were consecutively enrolled. The median age was 47 years (range, 27-72 years). Eighteen patients (36.0%) received weekly paclitaxel plus trastuzumab, 24 patients (48.0%) tri-weekly paclitaxel plus trastuzumab, and 8 patients (16.0%) tri-weekly docetaxel plus trastuzumab. Among 43 patients with measurable lesions, the response rate (RR) was 76.7%. The median follow-up duration was 29.2 months (range, 0.7-63.3 months). The median progression-free survival (PFS) was 17.6 months (95% confidence interval (CI), 13.4-21.9 months). The median overall survival (OS) was 47.0 months (95% CI, 35.3-58.6 months). The median serum amphiregulin level was 1.0 ng/mL with a maximum level of 4.4 ng/mL. Patients with high serum amphiregulin levels (≥0.5 ng/mL) had significantly shorter PFS (p=0.018) along with a tendency toward lower RR (p=0.237) and shorter OS (p=0.529) than the others. The in vitro colony forming assay demonstrated that the addition of amphiregulin resulted in increased proliferation of both SK-BR-3 and BT-474 cells. In addition, the anti-proliferative effect of trastuzumab was decreased in the presence of amphiregulin in both SK-BR-3 and BT-474 cells. The Western blot analysis showed that amphiregulin increased the phosphorylation of Akt and its downstream molecules in both SK-BR-3 and BT-474 cells. In addition, in the presence of amphiregulin, sustained phosphorylation of Akt and its downstream molecules was observed after trastuzumab treatment in both SK-BR-3 and BT-474 cells. Conclusions: High serum amphiregulin levels (≥0.5 ng/mL) predicted disease progression after first-line trastuzumab plus taxane chemotherapy in patients with HER2-positive metastatic breast cancer. Amphiregulin promoted the proliferation of HER2-positive breast cancer cells in vitro and induced trastuzumab resistance by activating PI3K/Akt pathway. Our results suggest that the measurement of serum amphiregulin levels by ELISA may provide additional information for the clinical outcome of trastuzumab-based chemotherapy in patients with HER2-positive breast cancer. Citation Format: Ji-Won Kim, Young Seok Joung, Ahrum Min, Hyun-Jin Nam, Jee Hyun Kim, Seock-Ah Im, Kyung-Hun Lee, Jin-Soo Kim, Tae-Yong Kim, Sae-Won Han, Yoon Kyung Jeon, Do-Youn Oh, Tae-You Kim, In Ae Park. Amphiregulin confers trastuzumab resistance by activating PI3K/Akt pathway in HER2-positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2386. doi:10.1158/1538-7445.AM2013-2386

Randomized phase II study of erlotinib (ERL) in two different schedules with concomitant modified XELOX in the first-line treatment of metastatic colorectal cancer (mCRC): Correlation with serial serum levels of amphiregulin (AMR) and transforming growth factor receptor-alpha (TGFa).

425 Background: This study evaluated the feasibility of combining XELOX with erlotinib in a 2 different schedules, and the prognostic significance of serum AMR and TGFa in previously untreated mCRC. Methods: Eligible patients (Pt) were randomized to the ‘continuous’ arm (Arm CON) [ERL 100mg daily D1-21, oxaliplatin (Ox) 130mg/m2 D1, capecitabine (Xe) 825mg/m2 bd D1-14, q3w], or the ‘intermittent’ arm (Arm INT) [ERL 150mg daily at alternate day on D 1-14, then 150mg daily on D15-21, Ox 130mg/m2 on D1, Xe 750mg/m2bd on D1-14, in a q3w]. Serum levels of AMR, TGFa and CEA were determined serially. KRAS mutation was determined from archived tumors. Results: 60 pts were randomized and there was no difference in the baseline characteristics between the 2 arms. Of the 58 pts evaluated for response, the overall response rates (ORR) were 56.3% (Arm CON) and 66.3% (Arm INT). At a median follow-up of 2.8 yrs, the median overall survival (OS) of pts in Arm CON and Arm INT were 9 m (95% CI: 7.2-18m) and 10.3m (95% CI: 7.2-15.6 m) respectively. KRAS mutation (n = 44 tumors) status did not predict ORR or survival. Gr 3-4 toxicities occurred in 53.3% (Arm CON) and 46.7% (Arm INT) of pts. Multivariate analysis showed that elevated baseline serum TGFa and AMR were independent predictors of inferior PFS and OS, respectively. Post-treatment drop in serum TGFa was associated with shorter OS and PFS, while a drop in serum TGFa and a rise in serum AMR were associated with inferior PFS and OS, respectively. Conclusions: Although there was no statistical difference in the toxicity and efficacy of the 2 arms, the ORR from XELOX and intermittent ERL compared favorably with historic control. KRAS mutation status was not predictive. Serum TGFa and AMR have prognostic significance in this cohort and further prospective studies are warranted. Clinical trial information: NCT01243047.

Direct and immune mediated antibody targeting of ERBB receptors in a colorectal cancer cell-line panel

A significant proportion of colorectal cancer (CRC) patients are resistant to anti-ERBB1 [avian erythroblastic leukemia viral (v-erb-b) oncogene homolog, receptor for EGF] monoclonal antibodies (Mabs). We evaluated both immune and nonimmune effects of cetuximab (anti-ERBB1 Mab), trastuzumab (anti-ERBB2 Mab), pertuzumab (anti-ERBB2 Mab), and lapatinib (dual ERBB1 and ERBB2 tyrosine kinase inhibitor) in a large well-characterized panel of 64 CRC cell lines to find response predictive tumor characteristics. There was a significant correlation between the direct effects of cetuximab and lapatinib. Both agents were associated (P = 0.0004) with "triple' wild-type status in KRAS, BRAF, and PIK3CA exon 20. Most cell lines were resistant to the direct effects of anti-ERBB2 Mabs, suggesting that the effects of lapatinib might mainly be through ERBB1. Microarray mRNA expression profiles of sensitive and resistant cell lines showed that although ERBB1 receptor or ligand levels did not associate with cetuximab sensitivity, high levels of ERBB2 (P = 0.036) and amphiregulin (P = 0.026) predicted sensitivity to lapatinib. However, higher ERBB1 expression predicted susceptibility to cetuximab-induced antibody-dependent cellular cytotoxicity and occurred independently of KRAS/BRAF/PIK3CA mutations (P = 0.69). Lapatinib may be an effective alternative therapy to cetuximab in triple wild-type tumors. Microarray analysis provides suggestive biomarkers for resistance. ERBB1 levels, independent of mutation status, predict immune killing. Therefore, anti-ERBB1 antibodies may be considered in CRC tumors with higher ERBB1 expression and favorable FcγR polymorphisms.

O-0007 Exploratory Serum Biomarker Analyses from BO21129, a Phase II Study of Erlotinib in Second-Line Pancreatic Cancer: Potential Role of Amphiregulin?

ABSTRACT Introduction Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor, is approved in combination with gemcitabine for advanced pancreatic cancer. As the use of biomarkers to guide treatment decisions for targeted therapies is an area of increasing interest, a post-approval commitment was made to investigate potential biomarkers for erlotinib in pancreatic cancer. This randomised, placebo-controlled, multicentre, phase II study examined whether any biomarkers were predictive of differential outcomes with erlotinib monotherapy in advanced pancreatic cancer. The primary data for this study have been presented previously and showed no correlation between any of the pre-specified biomarkers investigated and the primary endpoint of progression-free survival (PFS) with erlotinib. Here we present the results of exploratory analyses on serum biomarkers. Methods Serum samples were collected from patients at baseline, 3 weeks, and after treatment discontinuation. Randomisation was stratified by ECOG PS, region and smoking status, and the initial estimation of hazard ratio for the treatment effect was by unadjusted Cox regression. After observing imbalances in some baseline characteristics, additional post-hoc analyses using a stepwise model selection algorithm were performed to assess for any impact on PFS and OS. Serum biomarker analyses were conducted on amphiregulin, EGF, TGF-a, and sHer-2. Exploratory analyses of efficacy were performed in ‘low’ and ‘high’ subgroups, as compared to the median of each soluble marker in the overall population. Efficacy was analysed in subgroups using both the initial unadjusted analysis, as well as the post-hoc model that was developed in the overall population. Results Data on baseline serum markers were available for 199 out of 207 patients for amphiregulin and TGF-a, and 202 out of 207 patients for EGF and sHer-2. The adjusted post-hoc analysis of serum biomarkers showed that erlotinib provided a significant benefit in terms of PFS and OS in patients with high levels of amphiregulin (Table). Validation of results for other markers is ongoing and data will be presented. Conclusion Exploratory adjusted analyses of serum markers showed that patients with high serum amphiregulin concentrations achieved a significant benefit in both PFS and OS. The observation that patients with elevated baseline levels of amphiregulin obtained benefits in both PFS and OS with erlotinib may suggest potential for this biomarker in pancreatic cancer. Table 1 .

EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines

Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.