High IGF-1R Research Articles

Improved Immunohistochemical Detection of Type 1 Insulin-Like Growth Factor Receptor in Human Tumors

Background: Insulin-like growth factors (IGFs) are known to play important roles in cancer biology, prompting evaluation of drugs targeting type 1 IGF receptor (IGF-1R). However, there is considerable lack of consensus in immunohistochemical (IHC) studies of IGF-1R in human tumors, confounding attempts to assess the predictive and prognostic significance of IGF-1R expression and subcellular localization. Likely sources of variation include use of different IGF-1R polyclonal antibodies and methods for IHC. Here, we aimed to develop a robust IGF-1R IHC protocol using a monoclonal antibody, suitable for use in formalin-fixed paraffin-embedded (FFPE) tissues. Methods: Using controls including samples of FFPE tissues and tumor cells of defined IGF-1R expression, we used IHC and western blotting to compare polyclonal antibody #3027 with monoclonals #9750 and #14534 (Cell Signaling Technology). Results: Compared with #3027, the monoclonals exhibited superior discrimination between IGF-1R-high and IGF-1R-deficient cells in manual IHC, signal generated by #9750 reflecting differences in IGF-1R expression detected by western blotting. In tissues, IGF-1R detected by #14534 was predominantly plasma membrane-associated, while #9750 detected IGF-1R in the plasma membranes, cytoplasm and nucleus of prostate and renal cancers, recapitulating appearances we described using previous lots of #3027, and reflecting subcellular localizations reported using other techniques. Use of #9750 and #14534 in an autostainer showed adequate differentiation of high vs low IGF-1R cells, but did not recapitulate appearances of manually-stained tissues. We provide a detailed protocol for the preferred manual method using #9750. Conclusion: Standardization of IGF-1R IHC will promote understanding of the role of IGF-1R in tumor biology, and its potential as a candidate prognostic and predictive biomarker.

Association between the expression of IGF1R and estrogen receptor and efficacy of neoadjuvant chemotherapy in beast cancer patients

To detect the expression of IGF1R and estrogen receptor, and to explore the relationship between their expression and the pathological complete response (pCR) rate of neoadjuvant chemotherapy (docetaxel plus epirubicin) in breast cancer patients. We selected 139 women with breast cancer who underwent neoadjuvant chemotherapy (docetaxel plus epirubicin), and detected the expression of IGF1R and estrogen receptor in the samples taken before chemotherapy by Immunohistochemistry. The association between their expression and pCR rate of neoadjuvant chemotherapy was analyzed. Among the 139 cases, IGF1R was highly expressed in 45.3% (63/139) cases, and ER was positively expressed in 62.6% (87/139) cases. IGF1R was highly expressed in 54.0% (47/87) of the ER+ cases, significantly higher than that of ER- cases (30.8%, P<0.01). The overall pCR rate of all the 139 patients who received docetaxel plus epirubicin as neoadjuvant chemotherapy was 10.1% (14/139). The pCR rate was 19.2% (10/52) of the ER- patients and 4.6% (4/87) of the ER+ patients (P<0.05). The pCR rate was 10.5% (8/76) in the patients with low IGF1R expression and 9.5% (6/63) in the patients with high IGF1R expression (P>0.05). The patients with negative expression of ER and high expression of IGF1R showed the highest pCR rate (31.2%, P<0.01). Breast cancer patients with negative expression of ER and high expression of IGF1R are more sensitive to neoadjuvant chemotherapy of docetaxel plus epirubicin, and their pCR rate is significantly higher than that of other patients.

Clinical significance of IGF1R gene expression in patients with Stage II/III gastric cancer who receive curative surgery and adjuvant chemotherapy with S-1.

Curative resection and adjuvant chemotherapy is the standard treatment for Stage II/III gastric cancer, and S-1 is widely used for adjuvant chemotherapy. The type 1 insulin-like growth factor receptor (IGF1R) is involved in cell proliferation and prevention of apoptosis in many tumors. We evaluated the relative expression of the IGF1R gene to determine whether such expression correlates with outcomes in patients with Stage II/III gastric cancer. We measured the expression levels of the IGF1R gene in specimens of cancer and adjacent normal mucosa obtained from 134 patients with Stage II/III gastric cancer who received curative resection and adjuvant chemotherapy with S-1. We then evaluated whether the IGF1R gene expression levels correlate with clinicopathological characteristics and outcomes. IGF1R mRNA expression levels tended to be higher in cancer tissue than in the normal adjacent mucosa (P = 0.078). Multivariate analysis showed that high IGF1R gene expression was a significant independent predictor of poor survival in Stage II/III gastric cancer after curative resection and adjuvant chemotherapy with S-1 (HR 3.681, P = 0.007). The overall survival rate was significantly lower in patients with high IGF1R gene expression than in those with low expression (P = 0.012). IGF1R overexpression is considered a useful independent predictor of outcomes in Stage II/III gastric cancer after curative resection and adjuvant chemotherapy with S-1.

Combinational Therapy Enhances the Effects of Anti-IGF-1R mAb Figitumumab to Target Small Cell Lung Cancer.

BackgroundSmall cell lung cancer (SCLC) is a recalcitrant malignancy with distinct biologic properties. Antibody targeting therapy has been actively investigated as a new drug modality.MethodsWe tested the expression of IGF-1R and calculated the survival in 61 SCLC patients. We also evaluated the anti-tumor effects of anti-IGF-1R monoclonal antibody Figitumumab (CP) on SCLC, and tried two drug combinations to improve CP therapy.ResultsOur clinical data suggested that high IGF-1R expression was correlated with low SCLC patient survival. We then demonstrated the effect of CP was likely through IGF-1R blockage and down-regulation without IGF-1R auto-phosphorylation and PI3K/AKT activation. However, we observed elevated MEK/ERK activation upon CP treatment in SCLC cells, and this MEK/ERK activation was enhanced by ß-arrestin1 knockdown while attenuated by ß-arrestin2 knockdown. We found both MEK/ERK inhibitor and metformin could enhance CP treatment in SCLC cells. We further illustrated the additive effect of metformin was likely through promoting further IGF-1R down-regulation.ConclusionOur results highlighted the potential of anti-IGF-1R therapy and the adjuvant therapy strategy with either MEK/ERK inhibitor or metformin to target SCLC, warranting further studies.

IGF-1R expression is associated with HPV-negative status and adverse survival in head and neck squamous cell cancer.

Head and neck squamous cell carcinomas (HNSCC) are treated with surgery, radiotherapy and cisplatin-based chemotherapy, but survival from locally-advanced disease remains poor, particularly in patients whose tumors are negative for Human papillomavirus (HPV). Type 1 IGF receptor (IGF-1R) is known to promote tumorigenesis and resistance to cancer therapeutics. Here, we assessed IGF-1R immunohistochemistry on tissue microarrays containing 852 cores from 346 HNSCC patients with primary tumors in the oropharynx (n = 231), larynx (85), hypopharynx (28), oral cavity (2). Of these, 236 (68%) were HPV-negative, 110 (32%) positive. IGF-1R was detected in the cell membrane of 36% and cytoplasm of 92% of HNSCCs; in 64 cases with matched normal tonsillar epithelium, IGF-1R was overexpressed in the HNSCCs (P < 0.001). Overall survival (OS) and disease-specific survival (DSS) were reduced in patients whose tumors contained high membrane IGF-1R [OS: hazard ratio (HR) = 1.63, P = 0.006; DSS: HR = 1.63, P = 0.016], cytoplasmic IGF-1R (OS: HR = 1.58, P = 0.009; DSS: HR = 1.58, P = 0.024) and total IGF-1R (OS: HR = 2.02, P < 0.001; DSS: HR = 2.2, P < 0.001). High tumor IGF-1R showed significant association with high-tumor T-stage (P < 0.001) and HPV-negativity (P < 0.001), and was associated with shorter OS when considering patients with HPV-positive (P = 0.01) and negative (P = 0.006) tumors separately. IGF-1R was independently associated with survival in multivariate analysis including HPV, but not when lymphovascular invasion, perineural spread and T-stage were included. Of these factors, only IGF-1R can be manipulated; the association of IGF-1R with aggressive disease supports experimental incorporation of anti-IGF-1R agents into multimodality treatment programs for HPV-negative and high IGF-1R HPV-positive HNSCC.

Insulin growth factor 1 receptor expression is associated with NOTCH1 mutation, trisomy 12 and aggressive clinical course in chronic lymphocytic leukaemia.

IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL) patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540). High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high CD38 expression (p<0.0001), trisomy 12 (p<0.0001), and del(11)(q23) (p=0.014). Interestingly, higher IGF1R expression (p=0.002) characterized patients with NOTCH1 mutation (c.7541_7542delCT), identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation.

Receptor tyrosine kinase profiles and human papillomavirus status in oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinomas (OSCCs) are two distinct entities. We defined the molecular profiles of druggable receptor tyrosine kinases (RTKs) in both groups. E5 expression and RTK alterations were studied in 17 HPV-positive and 59 HPV-negative formalin-fixed OSCCs. RTK activation was explored in further 12 frozen OSCCs. The HPV-positive OSCCs showed E5 expression and 33.3% expressed low level of HER2. The HPV-negative OSCCs showed HER2 expression (31.2%), increased HER2 gene copy number (46.51%, P=0.045) and HER2 activation through HER2/EGFR heterodimerisation; HER3 (51.06%, P=0.008) and neuregulin (65.63%; P=0.03) expression, HER3 activation and HER3/EGFR heterodimerisation; and increased IGF-1R copy number (40.50%, P=0.021), high IGF-1R cDNA values (P= 0.002), IGF-1R activation and expression of IGF1/2 and amphiregulin. PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases. Human papillomavirus-positive and HPV-negative OSCC showed different RTK profiles. In HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRβ. In HPV-negative cases, HER3 may be a promising druggable biomarker that deserves further investigation. PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCC, particularly in HPV-positive/PI3KCA-mutated OSCCs because they may be driven by PI3KCA mutation alone.

Abstract 924: Predictive biomarker identification for combined anti-mTOR and anti-IGF-1R treatment in luminal B breast cancer

Abstract Luminal B breast cancer is one of the most aggressive subtypes of breast cancer for which effective treatments are needed. Recently, a phase I clinical study revealed that blockade of mTOR and IGF-1R has potential clinical activity in this cancer subtype. However, biomarkers to predict patient's response to the combination are still needed. Previous exploratory studies in ovarian cancer have associated low RAS-pathway activity, as determined by a RAS gene expression signature score, and high levels of IGF-1R axis genes with response to this combination therapy. In the present study we evaluated low RAS signature/high IGF-1/high IGF-1R as potential predictive biomarkers for anti-mTOR and anti-IGF-1R combination therapy in luminal B breast cancer patient-derived xenografts (PDX) treated with the allosteric mTOR inhibitor ridaforolimus and the monoclonal antibody against IGF-1R, dalotuzumab. Expression array analysis revealed that our 6 luminal B PDXs exhibited a low RAS signature score, compared to 6 HER2 and triple negative PDXs. Overall, luminal B PDXs expressed higher IGF-1R as compared to the other breast cancer subtypes with some variability among the different luminal models. Low expression of human IGF-pathway ligands IGF-1 and IGF-2 among the luminal PDXs, compared to luminal B breast cancers, suggested to us the need of exogenous IGF-1 ligand supplementation to derive IGF-1R feedback activation and combinatorial antitumor response. Accordingly, three high IGF-1R-PDXs treated with the anti-mTOR and anti-IGF-1R combination therapy supplemented with recombinant human IGF-1 exhibited statistically significant anti-tumor response as compared to single agents, whereas two low IGF-1R-expressing PDXs did not. As anticipated, ridaforolimus induced the IGF-1R-axis by increasing the levels of the adapter protein IRS-1, which potentiated Akt signaling. Hence, in vitro knockdown of IRS-1 promoted the antiproliferative activity of ridaforolimus in patient-derived tumor cells. In conclusion, our results demonstrate that baseline IGF-pathway expression, namely IGF-1, IGF-1R and IRS-1, predict benefit to combined anti-mTOR and anti-IGF-1R treatment in breast cancer and supports further exploration in luminal B breast cancers that derived benefit from this combination. Citation Format: Martin A. Rivas, Yasir H. Ibrahim, Olga Rodríguez, Pilar Antón, Patricia Cozar, Patricia Gómez-Pardo, Claudia Aura, Brian B. Haines, Sriram Sathyanarayanan, Theresa Zhang, Violeta Serra, José Baselga. Predictive biomarker identification for combined anti-mTOR and anti-IGF-1R treatment in luminal B 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 924. doi:10.1158/1538-7445.AM2014-924

Alterations of insulin-like growth factor-1 receptor gene copy number and protein expression are common in non-small cell lung cancer

AimsInsulin-like growth factor-1 receptor (IGF1R) is a tyrosine kinase membrane receptor involved in tumourigenesis that may be a potential therapeutic target. We aimed to investigate the incidence and prognostic significance...

High Coexpression of Both EGFR and IGF1R Correlates With Poor Patient Prognosis in Resected Non–Small-Cell Lung Cancer

BackgroundRecent experimental and biomarker evidence indicates that the epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF1R) interact in the pathogenesis of malignant epithelial tumors, including lung cancer. This study examines the expression of both receptors and their prognostic significance in surgically resected non–small-cell lung cancer (NSCLC). MethodsEGFR and IGF1R expression were evaluated in 184 patients with NSCLC (83 squamous cell carcinomas [SCCs], 83 adenocarcinomas [ADCs], and 18 other types) using immunohistochemical (IHC) analysis. Expression of both receptors was examined in matched fresh frozen normal and tumor tissues from 40 patients with NSCLC (20 SCCs and 20 ADCs) by Western blot analysis. ResultsHigh EGFR expression was detected in 51% of patients, and SCCs had higher EGFR expression than did non-SCCs (57.4% vs. 42.5%; P = .028). High IGF1R expression was observed in 53.8% of patients, with SCC having higher expression than non-SCC (62.6% vs. 37.3%; P = .0004). A significant association was shown between EGFR and IGF1R protein overexpression (P < .005). Patients with high expression of both receptors had a poorer overall survival (OS) (P = .04). Higher EGFR and IGF1R expression was detected in resected tumors relative to matched normal tissues (P = .0004 and P = .0009), with SCC having higher expression levels than ADC. ConclusionOur findings indicate a close interrelationship between EGFR and IGF1R. Coexpression of both receptors correlates with poor survival. This subset of patients may benefit from treatments cotargeting EGFR and IGF1R.

Dynamic and Nuclear Expression of PDGFRα and IGF-1R in Alveolar Rhabdomyosarcoma

Since the advent of tyrosine kinase inhibitors as targeted therapies in cancer, several receptor tyrosine kinases (RTK) have been identified as operationally important for disease progression. Rhabdomyosarcoma (RMS) is a malignancy in need of new treatment options; therefore, better understanding of the heterogeneity of RTKs would advance this goal. Here, alveolar RMS (aRMS) tumor cells derived from a transgenic mouse model expressing two such RTKs, platelet-derived growth factor (PDGFR)α and insulin-like growth factor (IGF)-1R, were investigated by fluorescence-activated cell sorting (FACS). Sorted subpopulations that were positive or negative for PDGFRα and IGF-1R dynamically altered their cell surface RTK expression profiles as early as the first cell division. Interestingly, a difference in total PDGFRα expression and nuclear IGF-1R expression was conserved in populations. Nuclear IGF-1R expression was greater than cytoplasmic IGF-1R in cells with initially high cell surface IGF-1R, and cells with high nuclear IGF-1R established tumors more efficiently in vivo. RNA interference-mediated silencing of IGF-1R in the subpopulation of cells initially harboring higher cell surface and total IGF-1R resulted in significantly reduced anchorage-independent colony formation as compared with cells with initially lower cell surface and total IGF-1R expression. Finally, in accordance with the findings observed in murine aRMS, human aRMS also had robust expression of nuclear IGF-1R. RTK expression status and subcellular localization dynamics are important considerations for personalized medicine.

Abstract B011: IGFR as a candidate therapeutic target for cancer-initiating cells in triple-negative breast cancer

Abstract Background: Cancer-initiating cells (CICs) play a crucial role in recurrence of triple-negative breast cancer (TNBC). Thus, therapies targeting CICs may help prevent TNBC recurrence. However, the molecular characteristics of TNBC CICs have not been fully elucidated. We sought to determine molecular traits of TNBC CICs by establishing a mouse model of TNBC that successfully recapitulates human TNBC. Methods: In TNBC, Rb and p53 pathways are frequently abrogated, and intracellular kinases such as Akt and MAPK are frequently activated. To recapitulate these genetic components, we used Ink4a/Arf-knockout (KO) mouse mammary epithelial cells (MECs). Ink4a/Arf transcribes p16Ink4a and p19Arf and activates Rb and p53; therefore, Ink4a/Arf-KO mimics abrogation of Rb and p53 pathways. In addition, we retrovirally introduced oncogenic HRAS(G12V), which is a robust activator of cytoplasmic kinases, including Akt and MAPK, into Ink4a/Arf-KO MECs in vitro. Mammospheres of HRAS transfected Ink4a/Arf-KO MECs were studied for expression patterns CD29, CD24, CD44, Sca1, CD61, and CD49f in tumor cells by FACS. Tumor cells were sorted according to their protein expression levels and inoculated into mammary fat pads of recipient mice to determine tumor-initiating activities of each subpopulation. The molecular profiles of CICs were determined by mouse microarray expression analysis. Results: Unexpectedly, CD49f-negative (CD49f-) tumor cells had much higher tumor-initiating activity than CD49f-positive (CD49f+) cells at 100-cell inoculations (CD49f-, 5/8 mice; CD49f+, 0/8 mice). We hypothesized that tumor initiation from CD49f- cells depends on their quiescent profile, which is a trait of CICs. To test this hypothesis, we further fractionated CD49f- cells into quiescent (CD49f-q) cells and dividing cells and inoculated these fractions into recipient mice. CD49f-q cells had higher tumor-initiating activity than CD49f- dividing cells. We concluded that CD49f-q cells were CICs in our model. Genomewide expressions of CD49f-q cells and CD49f+ cells were compared by paired t-test, and over- and underexpressed genes in CD49f-q cells were functionally annotated using ingenuity pathway analysis. One of the top-ranked bioprocesses of overexpressed genes in CD49f-q cells was the IGF-1 pathway. On the basis of these findings, we tested an IGF-1R/InsR dual kinase inhibitor in TNBC. We confirmed high IGF-1R and low InsR expression in TNBC cell lines (e.g., SUM149, MDA-MB-231, MDA-MB-468). With the IGF-1R/InsR dual kinase inhibitor, proportions of CICs, profiled as CD44+CD24-, were significantly reduced in a dose-dependent manner in SUM149 and MDA-MB-231 cells. Indeed, the dual kinase inhibitor dose-dependently inhibited in vitro mammosphere formation, a hallmark of CIC activity. An in vitro growth inhibition assay revealed that IGF-1R/InsR dual kinase inhibitor inhibited cell growth (all IC50 &amp;lt;0.5 μM), and an agar assay for colony formation confirmed this antitumor effect in TNBC cells. Conclusion: We found that CICs in TNBC function in part through the IGF-1 pathway. We found that an IGF-1R/InsR dual kinase inhibitor had significant antitumor effects via suppressing the human CIC phenotype. Citation Format: Naoto T. Ueno, Kazuharu Kai. IGFR as a candidate therapeutic target for cancer-initiating cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B011.

The Prognostic Role of Ephrin A2 and Endothelial Growth Factor Receptor Pathway Mediators in Patients With Advanced Colorectal Cancer Treated With Cetuximab

BackgroundPatients with colorectal cancer (CRC) with wild-type KRAS mutations are often treated with the endothelial growth factor receptor (EGFR) monoclonal antibody cetuximab. Despite the presence of a specific molecular target, most patients still do not derive benefit from this biological treatment. Our study explores the role of ephrin A2 (EphA2) receptor expression and of EGFR pathway mediators as predictors of cetuximab benefit. Patients and MethodsFormalin-fixed paraffin-embedded (FFPE) tumor biopsy samples from 226 cetuximab-treated patients with CRC were studied for mRNA expression of insulin growth factor binding protein 2 (IGFBP2), insulin growth factor receptor 1 (IGF1R), cMET, EphA2, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 by means of TaqMan reverse-transcribed polymerase chain reaction (RT-PCR). ResultsOf the 226 patients evaluable for exploratory analysis, 222 had complete data from follow-up visits. The univariate analysis revealed the following significant adverse prognostic factors for risk of death: high EphA2 mRNA levels (hazard ratio [HR], 1.61; P = .015), high HER2 mRNA levels (HR, 1.51; P = .045), and high IGF1R mRNA levels (HR, 1.56; P = .021). Low EphA2 tumor expression was significantly associated with objective response to cetuximab therapy. In multivariate analysis of a broad biomarker panel, factors with independent prognostic value included EphA2 mRNA levels (HR, 1.67; P = .029), high amphiregulin (AREG) mRNA levels in KRAS wild-type tumors (HR, 0.17; P < .0001), and high epiregulin (EREG) mRNA levels (HR, 0.38; P = .006). ConclusionHigh EphA2 receptor expression in CRC was associated with a worse outcome in patients treated with cetuximab-based therapy. Prospective validation in treated and control patients is required to dissect the predictive from prognostic role in advanced CRC.

Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple-negative breast cancer (TNBC): The immune and IGFR pathways.

1058 Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC definition by immunohistochemistry (IHC). In a phase II neoadjuvant clinical trial targeting EGFR in TNBC, we investigated various biomarkers to better identify an EGFR-sensitive population for potential further regimen development. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm (20-120); invasive ductal carcinoma: 96.7%; SBR grade III: 71.7%; complete pathological response (pCR) rate: 55.3% and 46.8% (according to Sataloff’s and Chevallier’s classifications, respectively). Paraffin-embedded and frozen tumor samples were collected before and after ST for biologic studies. Germinal BRCA1 mutations, and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs) were evaluated by IHC. Results: High CD8+ TILs was response-predictive (p=3.4.10-6). Tumor FOXP3 expression and high FOXP3 TILs tended to be predictive. High IGF-1R expressors responded better than low expressors (p = 0.028). Comparison of EGFR, IGF-1R and Her3 in biopsies versus surgical samples showed reduced EGFR levels in non-responders (p = 0.037), while Her3 (p = 0.049) and IGF-1R (p = 0.08) increased. Sequencing revealed BRCA1 mutations in 10% of pts. No difference of response (pCR) was observed between mutated patients and others. Somatic mutations of PI3K were observed in 6 pts. No mutations were observed in BRAF, KRAS, or EGFR. Conclusions: The CD8+ TIL count seems to predict the response to panitumumab. Tumor FOXP3 expression and high FOXP3 TILs also tended to be predictive. Tumor levels of IGF-1R seem to play a determinant role in TNBC response to anti-EGFR antibodies, in concordance with our observations in a head-and-neck cancer cohort. Clinical trial information: NCT00933517.

Abstract 4669: Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways.

Abstract Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC definition by immunohistochemistry (IHC) (Nielsen, 2004). In a phase II neoadjuvant clinical trial targeting EGFR in TNBC, we investigated various biomarkers to better identify an EGFR-sensitive population for potential further regimen development. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm (20-120); invasive ductal carcinoma: 96.7%; SBR grade III: 71.7%; complete pathological response (pCR) rate: 46.8% (Chevallier's classification). Paraffin-embedded and frozen tumor samples were collected before and after ST for biologic studies. Germinal BRCA1 mutations, and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs) were evaluated by IHC. Biopsies and surgical samples were analysed using Affimetrix arrays. Results : By univariate analysis, high CD8+ TILs was response-predictive (pCR rates: CD8 TILs: 84% high vs 10% low; p=3.4.10−6). Tumor FOXP3 expression and high FOXP3 TILs tended to be predictive (pCR rates: tumor FOXP3: 77% positive vs 36% negative, p=0.07; FOXP3 TILs: 66% high vs 37% low, p= 0.08). High IGF-1R expressors responded better than low expressors (80% vs 33%, respectively, p=0.028). As could be expected, a positive correlation between tumor FOXP3 expression and FOXP3 TILs was found (p = 2.7.10−4, r = 0.56). Surprisingly, a positive correlation was found between FOXP3 TILs and CD8+ TILs (p = 2.10−5, r = 0.59) and between tumor FOXP3 and CD8+ TILs (p = 6.3.10−3, r = 0.43). Comparison of EGFR, IGF-1R and Her3 in biopsies versus surgical samples showed reduced EGFR levels in non-responders (p = 0.037), while Her3 (p = 0.049) and IGF-1R (p = 0.08) increased. Sequencing revealed BRCA1 mutations in 10% of pts. No difference of response (pCR) was observed between mutated patients and others (p=0.91). Somatic mutations of PI3K were observed in 6 pts. No mutations were observed in BRAF, KRAS, or EGFR. Analysis of Affymetrix arrays for gene expression is underway and will be presented. Conclusions : Interestingly, the CD8+ TIL count seems to predict the response to panitumumab. Tumor FOXP3 expression and high FOXP3 TILs also tended to be predictive. Tumor levels of IGF-1R seem to play a determinant role in TNBC response to anti-EGFR antibodies, in concordance with our observations in a head-and-neck cancer cohort (Clin Canc Res 2012). Confirmatory and mechanistic studies of those biomarkers are warranted. Citation Format: Frederique Penault-llorca, Catherine Abrial, Marie-Melanie Dauplat, Maud Privat, Nancy Uhrhammer, Alexis Desrichard, Yannick Bidet, Nina Radosevic-Robin, Anne Cayre, Cecile Aube, Fabrice Kwiatkowski, Nassera Chalabi, Yves-Jean Bignon, Philippe Chollet, Jean-Marc Nabholtz. Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways. [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 4669. doi:10.1158/1538-7445.AM2013-4669

Abstract 85: Endocrine resistant luminal breast cancer xenografts are powerful models for the analysis of sensitivity to endocrine and everolimus treatments .

Abstract Background. Activation of the PI3K pathway is associated with resistance to endocrine treatments (ET) in luminal breast cancer (LBC). Everolimus, an mTOR inhibitor, has been approved in combination with exemestane in endocrine resistant breast tumors. However, no definite signature of ET resistance or predictive factors of sensitivity to everolimus (EVE) treatment have been evidenced so far. In order to further decipher these molecular patterns, we used patient derived LBC xenografts (LBC-PDX) treated with various ET and EVE based combinations. Methods. 3 LBC-PDX (named HBCx-3, HBCx-21 and HBCx-34) were treated with ET during several months to establish hormono-resistant (HR) xenografts. Both parental and HR models were treated by tamoxifen, estrogen deprivation, fulvestrant, EVE alone, and EVE combined with all three ET modalities. Tissue Micro Arrays from control and treated tumor samples were generated for IHC analyses. RT-PCR analyses were conducted on genes related to proliferation, ER, PI3K, IGF-1R and angiogenesis pathways. Results. In primary PDX, ET showed a poor level of sensitivity for HBCx-3 and a high and sustained efficacy of ER targeting and/or ER deprivation for HBCx-21 and HBCx-34 tumors. In vivo resistance to ET was confirmed in all HR variants with tumor growth rates faster than parental xenografts. ER expression was conserved in HR tumors, but expression of the ER-responsive genes PS2, PR and MYB was strongly decreased, indicating impaired ER transcriptional activity. Acquired resistance to tamoxifen and ovariectomy was also associated to a strong decrease of IGF-1R signaling in HBCx-21, and was related to higher P-AKT expression, although P-pS6 was highly expressed in both sensitive and resistant tumors. Furthermore, in vivo experiments showed that EVE alone was highly efficient in all models independently of P-AKT expression/PTEN loss. EVE combined with fulvestrant yielded the most complete and durable tumor growth inhibition. In HBCx-3, proliferation and pS6 levels were markedly decreased only by the EVE-fulvestrant combination. In HBCx-21 and HBCx-34 HR models, EVE-based treatments decreased proliferation, expression of angiogenesis markers, as well as P-pS6 expression with no variation in (P-)AKT nor (P-)mTOR levels. A high IGF-1R protein expression was found only in the HBCx-34 TAM-R tumor, which was strongly decreased in the fulvestrant-treated tumors. Conclusions. LBC-PDX with confirmed HR status have been established and may serve as models to study alternate therapies. Of note, in the HBCx-21 and -34 models, activation of the PI3K pathway is poorly correlated with baseline sensitivity to ET, suggesting other pathways of resistance. EVE alone is highly efficient in all settings, and combination with fulvestrant is promising at the in vivo and molecular levels. Proteomic, genomic and gene expression studies are ongoing. Citation Format: Paul H. Cottu, Thomas Bagarre, Jean-Jacques Fontaine, Franck Assayag, Sophie Richon, Sophie Chateau-Joubert, Aurélie Thuleau, Patricia de Cremoux, Khemaies Slimane, Didier Decaudin, Anne Vincent-Salomon, Ivan Bièche, Elisabetta Marangoni. Endocrine resistant luminal breast cancer xenografts are powerful models for the analysis of sensitivity to endocrine and everolimus treatments . [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 85. doi:10.1158/1538-7445.AM2013-85

Expression and Clinical Significance of mTOR in Surgically Resected Non-small Cell Lung Cancer Tissues: a Case Control Study

Mammalian target of rapamycin (mTOR) is master regulator of the PI3K/Akt/mTOR pathway and plays an important role in NSCLCs. Here we characterized mRNA and protein expression levels of mTOR and its functional associated molecules including PTEN, IGF-1R and 4EBP1 in surgically resected NSCLCs. Fifty-four patients with NSCLCs who underwent pulmonary resection were included in current study. mRNA levels of mTOR, PTEN, IGF-1R, and 4EBP1 were evaluated by RT-PCR and protein expression of mTOR, PTEN, and IGF-1R by immunohistochemistry (IHC). Association of expression of the relevant molecules with clinical characteristics, as well as correlations between mTOR and PTEN, 4EBP1 and IGF-1R were also assessed. The results of RT-PCR showed that in NSCLCs, the expression level of mTOR increased, while PTEN, 4EBP1 and IGF-1R decreased. Statistical analysis indicated high IGF-1R expression was correlated with advanced clinical stage (stage III) and PTEN expression was reversely associated with tumor size (P=0.16). The results of IHC showed mTOR positive staining in 51.8% of cases, while IGF-1R positive staining was found in 83.3% and loss of PTEN in 46.3%. Protein expression of mTOR was correlated with its regulators, PTEN and IGF-1R, to some extent. Abnormal activation of mTOR signaling, high expression of IGF-1R, and loss of PTEN were observed in resected NSCLC specimens. The poor expression agreement of mTOR with its regulators, PTEN, and IGF-1R, implied that combination strategy of mTOR inhibitors with other targets hold significant potential for NSCLC treatment.

Impact of insulin-like growth factor 1 receptor (IGF1R) and amphiregulin (AREG) expressions on survival in patients with stage II/III gastric cancer enrolled in the ACTS-GC study.

4012 Background: Exploratory biomarker analysis was conducted to identify factors related to outcomes of patients enrolled in the ACTS-GC study, a randomized controlled trial comparing adjuvant S-1 administration with surgery alone in 1,059 patients with stage II/III gastric cancer. Methods: Formalin-fixed paraffin-embedded surgical specimens were retrospectively examined in 829 patients (78.3%), and 63 genes involved in pyrimidine metabolic pathway, growth factor signaling pathway, apoptosis, DNA repair, etc., were analyzed by quantitative real-time RT-PCR after TaqMan assay-based pre-amplification. Gene expression levels were normalized to the geometric mean expressions of GAPDH, ACTB, and RPLP0, used as reference genes. The expression of each gene was categorized into low and high values at those median. The impacts of gene expression on survival were analyzed using the 5-year survival data of the ACTS-GC. The Benjamini and Hochberg procedure was used to control the false discovery rate (FDR). Results: Among 63 screened genes, IGF1R and AREG most strongly correlated with overall survival (OS), with FDR of 0.0048 and 0.018, respectively. OS was significantly worse in IGF1R high patients than in IGF1R low patients, but better in AREG high patients than in AREG low patients. The hazard ratio (HR) for death in the analysis of OS (S-1 vs. surgery alone) was lower in the high IGF1R group (HR, 0.55; 95%CI, 0.40-0.76) than in the low IGF1R group (HR, 0.72; 95%CI, 0.49-1.06). Likewise, the HR for death in the analysis of OS (S-1 vs. surgery alone) was much smaller in the low AREG group (HR, 0.57; 95%CI, 0.41-0.79) than in the high AREG group (HR, 0.74; 95%CI, 0.51-1.08). Interactions were not statistically significant. Conclusions: IGFR1 gene expression was associated with poor outcomes after curative resection of stage II/III gastric cancer, whereas AREG gene expression was associated with good outcomes. No interaction for survival was evident between S-1 and these gene expressions.

Molecular Characterisation of Long-Acting Insulin Analogues in Comparison with Human Insulin, IGF-1 and Insulin X10

Aims/HypothesisThere is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10.MethodsWe measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types.ResultsDetemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs.ConclusionsDetailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir.

Abstract 2866: Exploring the basis for platinum and ionizing radiation combination with R1507 (an anti-IGF1R monoclonal antibody) in a small cell lung cancer (SCLC) model using integrated functional and genomic approaches on both short- and long-term drug exposure reveals adaptive mechanisms

Abstract Introduction: R1507 has single agent activity and remarkable chemo- and radiosensitizing effect in defined SCLC cell lines in vitro. It exhibits a dramatic antitumoral effect when combined to IR and cisplatin in vivo. The molecular mechanisms underlying these effects remain unknown. Materials and methods: Three SCLC cell lines (H69, H146 and H526) were selected according to their specific mutational profile (H146 and H69 cells are mutated for MAP2K4 and both PIK3CA and C-MET, respectively). The effects of R1507 on IGF1R downstream signaling were explored using western blots. Both genomic (HG-U133 plus 2 microarray, Affymetrix) and classical functional analyses were performed on the following conditions: (A) R1507 alone short-term (ST-R1507) (3 hours) vs. long-term (LT-R1507) (4 weeks) exposure, (B) R1507 combined with cisplatin, (C) and with ionizing radiation (IR). Results: (A) ST-R1507 disrupts IGF1R signaling through Akt and MAPK pathways in a dose dependent manner in selected SCLC cell lines. High IGF-1R basal expression is not correlated with sensitivity to R1507. Only cell lines with IGF1R gene amplification (H526 and H146) exhibit sensitivity to R1507. The addition of either MK-2206 (Akt inhibitor) or PHA665752 (c-Met tyrosine kinase inhibitor) to R1507 does not restore the sensitivity to IGF-1R inhibition in H69 cells. The addition of U0126 (MEK inhibitor) to R1507 does not restore the sensitivity to IGF-1R inhibition in H146 cells. LT- vs. ST-R1507 analyses reveal evidence of adaptive mechanisms susceptible to promote tumor escape to R1507 as single agent: (i) activation of cell-cell adhesion, beta-arrestin and MAPK gene expression pathways, (ii) activation of multiple cell surface receptors and intracellular proteins implicated in proliferation, angiogenesis and survival, (iii) transient reduction of phosphorylated IGF-1R staining intensity, in H526 xenografts in vivo. Regarding combination regimens, we identified: (B) the apoptotic mitochondrial and the NER pathways as majorly sensitizing the effects of LT- R1507 to Cisplatin, and (C) the TGF-beta pathway as the key mediator of the sensitization of LT-R1507 to IR. Conclusion: Combined genomic and functional approaches performed under LT-R1507 exposure unravel the activation of multiple alternative signaling pathways that are expected to promote tumor escape. Our study also provides key biological basis for translating the R1507 combined with Cisplatin and IR in the SCLC clinical setting. To analyze both short- and long-term drug exposure appears critical when evaluating new antitumoral regimens. 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 2866. doi:1538-7445.AM2012-2866