Antonios Danikas

Epidermal Growth Factor Receptor (EGFR) Is Overexpressed in High-Grade Dysplasia and Adenocarcinoma of the Esophagus and May Represent a Biomarker of Histological Progression in Barrett's Esophagus (BE)

OBJECTIVES:The assessment of cancer risk in patients with Barretts esophagus (BE) is currently fraught with difficulty. The current gold standard method of assessing cancer risk is histological assessment, with the appearance of high-grade dysplasia (HGD) as the key event monitored. Sampling error during endoscopy limits the usefulness of this approach, and there has been much recent interest in supplementing histological assessment with molecular markers, which may aid in patient stratification.METHODS:No molecular marker has been yet validated to accurately correlate with esophageal histological progression. Here, we assessed the suitability of several membranous proteins as biomarkers by correlating their abundance with histological progression. In all, 107 patient samples, from 100 patients, were arranged on a tissue microarray (TMA) and represented the various stages of histological progression in BE. This TMA was probed with antibodies for eight receptor proteins (mostly membranous).RESULTS:Epidermal growth factor receptor (EGFR) staining was found to be the most promising biomarker identified with clear increases in staining accompanying histological progression. Further, immunohistochemistry was performed using the full-tissue sections from BE, HGD, and adenocarcinoma tissues, which confirmed the stepwise increase in EGFR abundance. Using a robust H-score analysis, EGFR abundance was shown to increase 13-fold in the adenocarcinoma tissues compared to the BE tissues. EGFR was “overexpressed” in 35% of HGD specimens and 80% of adenocarcinoma specimens when using the H-score of the BE patients (plus 3 s.d.) as the threshold to define overexpression. EGFR staining was also noted to be higher in BE tissues adjacent to HGD/adenocarcinoma. Western blotting, although showing more EGFR protein in the adenocarcinomas compared to the BE tissue, was highly variable. EGFR overexpression was accompanied by aneuploidy (gain) of chromosome 7, plus amplification of the EGFR locus. Finally, the bile acid deoxycholic acid (DCA) (at neutral and acidic pH) and acid alone was capable of upregulating EGFR mRNA in vitro, and in the case of neutral pH DCA, this was NF-κB dependent.CONCLUSIONS:EGFR is overexpressed during the histological progression in BE tissues and hence may be useful as a biomarker of histological progression. Furthermore, as EGFR is a membranous protein expressed on the luminal surface of the esophageal mucosa, it may also be a useful target for biopsy guidance during endoscopy.

Three Methods for 18F Labeling of the HER2-Binding Affibody Molecule ZHER2:2891 Including Preclinical Assessment

Human epidermal growth factor receptor (HER2)–targeted Affibody molecules radiolabeled with 18F allow the noninvasive assessment of HER2 status in vivo through PET imaging. Such agents have the potential to improve patient management by selecting individuals for HER2-targeted therapies and allowing therapy monitoring. The aim of this study was to assess different 18F radiolabeling strategies of the HER2-specific Affibody molecule ZHER2:2891, preclinically determine the biologic efficacy of the different radiolabel molecules, and select a preferred radiolabeling strategy to progress for automated manufacture. Methods: Cysteine was added to the C terminus of the Affibody molecule for the coupling of maleimide linkers, and 3 radiolabeling strategies were assessed: silicon-fluoride acceptor approach (18F-SiFA), 18F-AlF-NOTA, and 4-18F-fluorobenzaldehyde (18F-FBA). The biodistributions of the radiolabeled Affibody molecules were then determined in naïve CD-1 nude mice, and tumor targeting was assessed in CD-1 nude mice bearing high-HER2-expressing NCI-N87 tumors and low-HER2-expressing A431 tumors. The 111In-ABY-025 compound, which has demonstrable clinical utility, served as a reference tracer. Results: The non–decay-corrected radiochemical yields based on starting 18F-fluoride using the 18F-FBA, 18F-SiFA, and 18F-AlF-NOTA methods were 13% ± 3% (n = 5), 38% ± 2% (n = 3), and 11% ± 4% (n = 6), respectively. In naïve mice, both the 18F-AlF-NOTA-ZHER2:2891 and the 111In-ABY-025 compounds showed a significant kidney retention (70.3 ± 1.3 and 73.8 ± 3.0 percentage injected dose [%ID], respectively, at 90 min after injection), which was not observed for 18F-FBA-ZHER2:2891 or 18F-SiFA-ZHER2:2891 (4.8 ± 0.6 and 10.1 ± 0.7 %ID, respectively, at 90 min). The 18F-SiFA-ZHER2:2891 conjugate was compromised by increasing bone retention over time (5.3 ± 1.0 %ID/g at 90 min after injection), indicating defluorination. All the radiolabeled Affibody molecules assessed showed significantly higher retention in NCI-N87 tumors than A431 tumors at all time points (P < 0.05), and PET/CT imaging of 18F-FBA-ZHER2:2891 in a dual NCI-N87/A431 xenograft model demonstrated high tumor-to-background contrast for NCI-N87 tumors. Conclusion: The HER2 Affibody molecule ZHER2:2891 has been site-selectively radiolabeled by three 18F conjugation methods. Preliminary biologic data have identified 18F-FBA-ZHER2:2891 (also known as GE226) as a favored candidate for further development and radiochemistry automation.

Abstract 4294: GE152: In vivo detection of tumor apoptosis as a tool for assessment of therapeutic efficacy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL A major challenge in personalized healthcare is predicting how effective a drug treatment is. This is particularly the case in oncology, where there is a call for better therapy monitoring to maximize drug treatment effectiveness leading to improved patient survival and help reduce healthcare costs. GE152, a nuclear imaging agent under development at GE Healthcare, is being evaluated preclinically as a tool to assess therapeutic efficacy by detecting tumor apoptosis. GE152 is based on a 99mTechnetium radiolabelled peptide that shows nanomolar affinity for a specific cell death target, as demonstrated by studies using Biacore technology. Biodistribution using the murine lymphoma (EL4) tumour therapy model has shown increased tumor uptake and retention of GE152 following chemotherapy, with positive tumor:muscle and tumor:blood ratios. Correlation of tumor apoptosis levels (determined by caspase activity) with GE152 tumor retention suggest a trend of increasing agent retention with rising levels of apoptosis (GE152 retention in low apoptotic tumors is 4.9%ID/g; GE152 retention in high apoptotic tumors is 8.2%ID/g).Further validation of GE152 was carried out using an apoptosis-specific inducible cell death model whereby HT29 colorectal cancer cell xenografts engineered to inducibly express either a constitutively active form of caspase-3 (which causes synchronous cell death in vivo when exposed to doxycycline) or an inactive point mutant. GE152 demonstrated greater uptake in tumors undergoing apoptosis (3.6% ID/g) than in controls (1.2% ID/g), correlating with caspase activity levels (and subsequent apoptosis) as determined both enzymatically and by IHC and blood-borne biomarkers of cell death. These results are in agreement with preliminary preclinical imaging studies using SPECT/CT, where region of interest analysis has demonstrated increased post-therapy tumor retention of GE152. We are currently optimizing the performance of GE152 by assessing different radiolabelling precursors to improve imaging agent pharmacokinetics. In addition to the current 99mTechnetium-based approach, we are exploring 18F radiolabelling options that would allow expansion of the agents utility to PET imaging. 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 4294. doi:1538-7445.AM2012-4294

Reply: Al18F Labeling of Affibody Molecules

TO THE EDITOR: Glaser et al. recently described the labeling of F-ZHER2:2891-Cys-NOTA-(COOH)2-AlF (18F-12) (1) and compared it in vivo to the biodistribution of that Affibody (Affibody AB) with 18F attached to carbon and silicon, as well as an 111In-DOTA-Affibody. They reported that the Al18F-labeled Affibody had a similar biodistribution to the 111In-Affibody, as previously noted by Heskamp et al. (2), and also observed that the Al18F-labeled Affibody had high uptake and retention in the kidney (;80 percentage injected dose [%ID], like the 111In-Affibody). This is presumably because the small-sized Affibody is eliminated through the kidneys, where it is rapidly catabolized, with the resulting Al18F complex residualized in the renal tubules in the same manner as the 111In-DOTA complex (3). In contrast, when the carbonand silicon-labeled Affibody molecules are metabolized in the kidney, the 18F-labeled metabolites are eliminated from the kidney cells, greatly reducing renal uptake. Although this clearly serves as an advantage for this product, much like differences between radioiodinated and radiometal-labeled antibody fragments, it is important to emphasize that renal uptake of the Al18F-Affibody product is a property of the Affibody targeting agent and not the Al18F complex. Previous studies with our pretargeting peptide (4) and the Al18F-NOTA-pegylated arginine-glycineaspartic acid dimer (PRGD2) peptide (5) both showed excellent renal clearance in the mouse models, and the Al18F-NOTAPRGD2 peptide also had good renal clearance in humans (6). It should also be noted that the 18F-Affibody labeled through a carbon atom had high hepatobiliary clearance (40–50 %ID in the intestines), whereas the Al18F-labeled Affibody had low uptake in the intestines. The high hepatobiliary accretion might be considered at least as undesirable as the high renal retention, depending on the use of the agent. Glaser et al. also reported a 2-fold lower labeling yield for their Al18F-Affibody than the Al18F-labeling yield of a similar Affibody bearing the same NOTA ligand (11% vs. 21%), and this despite the fact that Heskamp et al. used a lower amount of the Affibody (2). Although we cannot discount the possibility that slight differences in the Affibody structure could have influenced the yields, we strongly suspect the yield differences are attributable to the lack of a co-solvent in the labeling procedure used by Glaser et al. Indeed, we have shown that the use of a co-solvent generally improves yields 2-fold (7). Thus, we believe it is important when comparing labeling technologies to attempt to optimize or normalize each procedure, or if not empirically assessed, to state the conditions that might have affected yields when this information has been published previously. Second, whereas the nonresidualizing 18F-linkage used by Glaser et al. provided lower renal uptake, there likely are other situations, such as in target cells with a more rapid metabolism, in which a residualizing form of 18F afforded by the AlF method would be preferred (8). DISCLOSURE

Abstract 362: [18F]-FLT Positron Emission Tomography can be used to image the response of sensitive tumours to PI3-Kinase inhibition with the novel agent GDC-0941.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Emerging targeted therapeutics are characterised by high efficacy rates in selected patient sub-populations as well as potentially high running costs. This calls for companion diagnostic tools that contribute to the better management of such therapies, in terms of patient selection and monitoring therapeutic response. The Phosphatidylinositide 3-kinase pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the PET tracer [18F]-FLT is suitable to mark the effect of the novel PI-3K inhibitor GDC-0941 which has entered Phase II clinical trial. Methods Nude mice bearing U87 and HCT116 xenografts were imaged at baseline with [18F]-FLT (10-15MBq per mouse produced using a GE Tracerlab). GDC-0941 or vehicle was then administered at a concentration of 50mg/kg twice daily by gavage and animals were rescanned at acute (∼18h) and chronic (∼186h) timepoints post-therapy. Standard uptake value (SUV)s for tumour uptake were calculated, and tissue was analysed at sacrifice for phospho-AKT. To generate normalised uptake values (NUV)s the SUV [tumour] was divided by the SUV for heart. NUV figures are given for the 100-105min post-injection time interval. Results Growth of U87 xenografts was significantly inhibited by GDC-0941 treatment throughout the course of the study (average growth rate of treated = –1.0±9.3 mm3, control = 69.2±22.1mm3 per day). Growth of HCT116 xenografts was not significantly different between groups (average growth rate of treated = 34.6±15.9 mm3, control =54.8±11.1 mm3 per day). Tumour uptake of [18F]-FLT was significantly reduced in treated animals bearing U87 xenografts at the acute timepoint compared to baseline (NUVmax 2.17 ± 0.38 vs 1.59 ± 0.29, p<0.01), whereas uptake in HCT116 tumours was unchanged. Analysis of the downstream marker phospho-AKT showed that this was decreased in U87, but not HCT116, tumours. Conclusion [18F]-FLT is a strong candidate for the non-invasive measurement of GDC-0941 action. In addition to FLT, we are assessing the performance of a range of other imaging agents, including proprietary tracers, that can potentially bring value in monitoring of PI3-kinase targeting therapies. 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 362. doi:1538-7445.AM2012-362

Abstract 353: In vivo PET imaging of HER2 expression with GE226: An 18F-labelled affibody molecule

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The assessment of HER2 expression in biopsies from primary lesions of breast cancer patients is a standard procedure to select patients for HER2-targeted therapies. However, in metastatic disease in which HER2 status can change, determination of HER2 expression is not standard procedure, which complicates patient management. Herein we report evidence for GE226 ([18F]FBA-ZHER2:2891), a highly specific HER2 targeted imaging agent that can determine HER2 expression levels in preclinical tumour models. We propose that GE226 can be progressed to clinical development for non-invasive determination of the HER2 status in recurrent breast cancer patients to improve the clinical management and therapy selection. Methods: The highly selective HER2 targeted 18F-labelled Affibody molecule GE226 was characterized in a murine dual tumour breast cancer model bearing NCI-N87 (high HER2 status) and A431 (low HER2 status) xenografts in separate flanks. Tumour-bearing mice were injected with 3 to 10 MBq of GE226, followed by biodistribution or positron emission tomography (PET) imaging analysis. Results: Biodistribution analysis demonstrated good differentiation of GE226 retention between high and low HER2 expressing tumours (8.4% ID/g and 3.4% ID/g respectively, 30 min post injection). GE226 cleared quickly from background tissue, including kidneys, with excellent ratios for tumour-to-muscle (8.9 for high HER2 status tumours and 3.6 for low HER2 status tumours, 30 min post injection) and tumour-to-blood (2.5 for high HER2 status tumours and 1.0 for low HER2 status tumours, 30 min post injection). PET imaging of GE226 in the dual tumour mouse model showed a marked difference in signal intensity between the two tumour types. Conclusions: The highly selective HER2 targeted Affibody molecule GE226 can image different levels of HER2 expression in a dual-tumour preclinical model of breast cancer with good target-to-background ratios. These data compare favourably with previous patient SPECT and PET studies using the 111In- or 68Ga-labelled HER2-binding Affibody molecule ABY-002 (Baum et al., J Nucl Med. 2010;51(6):892-7) which supports the efficacy of this class of Affibody tracer for visualization of HER2 expressing metastases. We plan to progress GE226 further to assess HER2 status in metastatic breast cancer patients in clinical PET studies. 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 353. doi:1538-7445.AM2012-353