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Representative 3D-rendering of segmented MCF-7, MDA-MB-468 and MDA-MB-231 xenografts following PET/CT image acquisition 3 h after 89Zr-DFO-ZHER3:8698 injection.

Purpose: Recent studies have highlighted a role of HER3 in HER2-driven cancers (e.g., breast cancer), implicating the upregulation of the receptor in resistance to HER-targeted therapies and Hsp90 inhibitors (e.g., AUY922). Therefore, we have developed an affibody-based PET radioconjugate that quantitatively assesses HER3 changes induced by Hsp90 inhibition in vivo.Experimental Design: ZHER3:8698 affibody molecules were conjugated via the C-terminus cysteine to DFO-maleimide for 89Zr radiolabeling. The probe was characterized in vitro and in vivo in a panel of human breast cell lines and xenograft models with varying HER3 receptor levels. In addition, the radioconjugate was investigated as a tool to monitor the outcome of AUY922, an Hsp90 inhibitor, in an MCF-7 xenograft model.Results: We demonstrated that 89Zr-DFO-ZHER3:8698 can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our in vitro findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to treatment with AUY922 through HER3/IGF-1Rβ–mediated signaling. Of note, the lack of response in vitro due to HER3 recovery was confirmed in vivo using 89Zr-DFO-ZHER3:8698–based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 upregulation concomitant with an increase in IGF-1Rβ expression.Conclusions: These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not responding to targeted therapies due to HER3 recovery. Clin Cancer Res; 24(8); 1853–65. ©2018 AACR.

Materials and Methods Fig. S1. Time course of AUY922 treatment in BT-474 and MCF-7 cells. Fig. S2. Effects of AUY922 and PPP treatment in low and high HER2-expressing breast cancer cell lines. Fig. S3. Effect of HRG stimulation in AUY922 and PPP treated MCF-7 cells. Fig. S4. AUY922 and PPP treatment effect on the proliferative and invasive phenotype of BT-474 and MCF-7 breast cancer cells. Fig. S5. HPLC chromatograms of purified DFO-affibody conjugates. Fig. S6. MALDI-MS spectra of purified DFO-ZHER3:8698 (A) and DFO-ZTAQ (B). Fig. S7. Representative radio-ITLC of the crude radiolabelling mixture of 89Zr-DFO-ZHER3:8698. Fig. S8. Representative radio-ITLC of the 89Zr-DFO-ZHER3:8698 serum stability at 0, 3 and 24 h. Fig. S9. In vivo 89Zr-DFO-ZHER3:8698 tumour uptake quantification by image analysis. Fig. S10. Ex vivo biodistribution of 89Zr-DFO-ZHER3:8698 following AUY922 treatment in MCF-7 xenografts. Fig. S11. Microvessel density analysis in response to AUY922 treatment in MCF-7 xenografts. Fig. S12. Tumour volume estimation in response to AUY922 treatment in MCF-7 xenografts. Fig. S13. AUY922 treatment response monitoring in BT-474 xenografts by 89Zr-DFO-ZHER3:8698 PET/CT imaging. Fig. S14. Texture analysis of 89Zr-DFO-ZHER3:8698 PET imaging data following AUY922 treatment in MCF-7 xenografts. Fig. S15. Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and HER3 protein expression per control and AUY922-treated MCF-7 xenografts. Fig. S16. HER3 intra-tumoral heterogeneity highlighted by histopathological analysis of control and AUY922-treated MCF-7 xenografts. Fig. S17. HER2-HER3 interaction in BT-474 and MCF-7 cells following AUY922 treatment. Fig. S18. Scheme illustrating the proposed effect of AUY922-induced Hsp90 inhibition in MCF-7 breast cancer xenografts. Table S1. Densitometric analysis of Western blots related to the treatment of BT-474, MCF-7, MCF-7 HER2+++ (-dox), and MCF-7 HER2+ (+dox) following 48 h treatment with AUY922 and PPP alone, or in combination. Table S2. Biodistribution results for 89Zr-DFO-ZHER3:8698 and 89Zr-DFO-ZTAQ (3 µg, 7.2-8.1 MBq/mouse) at 3 h after injection. Table S3. Biodistribution results for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S4. Tumour/organ ratios for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S5. HER3 and IGF1R co-occurrence in breast invasive carcinoma samples. Table S6. HER3 and IGF1R alterations lead to a shorter overall survival in breast invasive carcinoma samples. Table S7. HER3 and IGF1R alterations lead to a shorter disease free survival in breast invasive carcinoma samples. Table S8. HER3 and IGF1R co-occurrence in HER2 up-regulated breast invasive carcinoma samples.

Behçet’s disease (BD) is a multisystem inflammatory disorder of unknown etiology, characterized by oral and genital ulceration, with other complications including eye, skin, joint, and central nervous system (CNS) lesions. Diagnosis is based on clinical findings, which may differ between patients. There is a strong genetic basis for BD; however, only a few genes have been associated with the disease across the geographical spread of BD. In this article, we discuss the history and combination of genes involved in this complex disease in relation to the geographical range and present our view that the disease has developed from a Darwinian perspective, with different gene polymorphisms that affect the same biological pathway. Moreover, these mutations individually are protective mechanisms against the disease relevant to each region, which affected both archaic and modern humans.