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Format: .mp4; Size: 59 MB.Description of Movie Many rapidly dividing tumor cells express or overexpress surface receptor molecules that can be targeted with antibodies. Tracers (for example, the radionuclide zirconium-89) can be attached to these antibodies and administered to patients. Once the antibody reaches the surface receptor on the tumor cell, it is internalized and digested in an endosome, releasing the tracer. Accumulation of the tracer in the tumor cells can then be visualized.NOTE: There is no audio component associated with this video file.

Supplementary Figure 1. The arithmetic mean concentration-time profiles for AUY922 and BJP762. Supplementary Table 1. The most common adverse events of AUY922. Supplementary Table 2. Adverse events with CTCAE grades 3 and 4. Supplementary Table 3. The SUVmax of 89Zr-trastuzumab before and during treatment with AUY922. Supplementary Table 4. SUVmax of 89Zr-bevacizumab before and during treatment with AUY922.

In vivo quantification of ��Zr-CX-072, ��Zr-PbCtrl and ��Zr-CX-075 PET imaging.

Purpose: AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 (89Zr) or fluorescent dye to evaluate the tumor-targeting properties.Experimental Design: 89Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μg 89Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, 89Zr-Mec14, served as a negative control. 89Zr-AMG211 integrity was determined in tumor lysates ex vivo. Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, 89Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199.Results: 89Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μg 89Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of 89Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. 89Zr-Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured 89Zr-AMG211 fulfilled release specifications.Conclusions: 89Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior. Clin Cancer Res; 24(20); 4988–96. ©2018 AACR.

Purpose: To provide proof of principle of safety, breast tumor–specific uptake, and positive tumor margin assessment of the systemically administered near-infrared fluorescent tracer bevacizumab–IRDye800CW targeting VEGF-A in patients with breast cancer.Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab–IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and coregistration of tumor tissue and healthy tissue.Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared with those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level.Conclusions: Our study shows that systemic administration of the bevacizumab–IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor margin uptake as evaluated by a systematic validation methodology. The findings are a step toward a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in the tumor tissue. Clin Cancer Res; 23(11); 2730–41. ©2016 AACR.

Figure legends for Figure S1-S6.

Supplemental Materials and Methods; Suppl. Fig. 1. Comparison of tumor vs. skin fluorescence obtained from human breast tissue specimen; Suppl. Fig 2: Magnified comparison between fluorescence images obtained from 3mm thick paraffin slices and 4microm paraffin slices.

Purpose: The mTOR pathway is frequently activated in ovarian cancers. mTOR inhibitors, such as everolimus, can reduce VEGF-A production by cancer cells. We investigated whether early everolimus treatment effects could be monitored by positron emission tomography (PET) with 89Zr-bevacizumab.Experimental Design: The effect of everolimus on VEGF-A secretion was determined in a panel of human ovarian cancer cell lines and in A2780luc+ ovarian cancer cells xenografted subcutaneously in BALB/c mice. Mice received daily 10 mg/kg everolimus intraperitoneally (i.p.) for 14 days. PET scans with the tracer 89Zr-labeled bevacizumab were conducted before and after treatment. Ex vivo89Zr-bevacizumab biodistribution and correlative tissue analyses were conducted. Tumor VEGF-A levels were measured with ELISA and mean vascular density (MVD) was determined with immunohistochemistry.Results: Everolimus treatment reduced VEGF-A levels in the supernatant of all cell lines. Everolimus lowered 89Zr-bevacizumab tumor uptake by 21.7% ± 4.0% [mean standardized uptake value (SUVmean) 2.3 ± 0.2 vs. 2.9 ± 0.2, P < 0.01]. Ex vivo biodistribution also showed lower tracer uptake in the tumors of treated as compared with control animals (7.8 ± 0.8%ID/g vs. 14.0 ± 1.7%ID/g, P < 0.01), whereas no differences were observed for other tissues. This coincided with lower VEGF-A protein levels in tumor lysates in treated versus untreated tumors (P = 0.04) and reduced MVD (P < 0.01).Conclusion: Tumor VEGF-A levels are decreased by everolimus. 89Zr-bevacizumab PET could be used to monitor tumor VEGF-A levels as an early biomarker of the antiangiogenic effect of mTOR inhibitor therapy. Clin Cancer Res; 18(22); 6306–14. ©2012 AACR.

Supplementary Table S2 from Involvement of the TGF-β and β-Catenin Pathways in Pelvic Lymph Node Metastasis in Early-Stage Cervical Cancer

Supplementary Figures S1-S2 from Involvement of the TGF-β and β-Catenin Pathways in Pelvic Lymph Node Metastasis in Early-Stage Cervical Cancer

Near-Infrared Optical Imaging of Positive Tumor Margin.

PDF file - 20K, Schematic representation of the study design

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and nonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623–31. ©2016 AACR.

Co-localization of Bevacizumab-IRDye800CW, hematoxylin / eosin (H/E), VEGF-A, Collagen and CD34 in Ductal Carcinoma In Situ (DCIS).

Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer.Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N0) and 19 with positive lymph nodes (N+), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways.Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N0, and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N+ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N0 and N+ tumors (P < 0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N0 (OR: 0.20, 95% CI: 0.06–0.66) and the β-catenin pathway (p120 positivity) to N+ (OR: 1.79, 95%CI: 1.05–3.05).Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated noncanonical β-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer. Clin Cancer Res; 17(6); 1317–30. ©2011 AACR.

A. PET uptake in the circulation per cohort, as determined by region of interest drawn in left ventricle; cohort 1 (in blue) contained 2 patients (0 mg additional MMOT0530A), cohort 2 (in green) contained 9 patients (10 mg additional MMOT0530A). On X axis the different PET moments: 2, 4 and 7 days post injection. On the Y axis the mean SUVmax per cohort.

Supplementary Tables

Co-localization of Bevacizumab-IRDye800CW, hematoxylin/eosin (H/E), VEGF-A, Collagen and CD34 in Breast Cancer and a Satellite Lesion.

Purpose: To evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of mapatumumab, a fully human monoclonal antibody targeting tumor necrosis factor–related apoptosis–inducing ligand receptor 1 (TRAIL-R1), in combination with gemcitabine and cisplatin.Experimental Design: Patients with advanced solid tumors received gemcitabine 1,250 mg/m2 i.v. on days 1 and 8 and cisplatin 80 mg/m2 i.v. on day 1 of each 21-day cycle. Escalating mapatumumab doses were administered i.v. every 21 days. Toxicity was evaluated and pharmacokinetic analysis of plasma mapatumumab, gemcitabine, 2-difluoro-2-deoxyuridine, and unbound and total platinum was done. TRAIL-R1 tumor expression was determined immunohistochemically.Results: Forty-nine patients received mapatumumab (1 mg/kg, n = 4; 3 mg/kg, n = 7; 10 mg/kg, n = 12; 20 mg/kg, n = 13; or 30 mg/kg, n = 13). A median of six cycles (range, 1-48) was administered. The adverse events most commonly observed reflect the toxicity profile of gemcitabine and cisplatin. Dose-limiting toxicities were seen in 3 of 12 patients at 10 mg/kg, consisting of grade 3 transaminitis, neutropenic fever, and grade 4 thrombocytopenia. At 20 mg/kg, 2 of 12 patients had dose-limiting toxicities, including grade 4 thrombocytopenia and grade 4 fatigue. The maximum tolerated dose was not reached. Pharmacokinetic interactions have not been observed. Twelve patients had a partial response, and 25 patients showed stable disease with a median duration of 6 months.Conclusions: Mapatumumab in combination with gemcitabine and cisplatin is safe and well tolerated at doses up to 30 mg/kg. Further studies on this combination are warranted. (Clin Cancer Res 2009;15(17):5584–90)

Supplementary Figures S1-S4 and Supplementary Table S1 including Supplementary Figure legend and Supplementary Table caption Figure S1. Flow chart of the drug substance (N-sucDf-AMG211) manufacturing process and drug product (89Zr-AMG211) formulation and filling process. Figure S2. Quality control of 89Zr AMG211. Representative size exclusion high performance liquid chromatography chromatogram of 89Zr AMG211 with 280 nm signal (top panel), radiochemical signal (middle panel) and the overlay (lower panel). Figure S3. Immunoreactivity of 89Zr AMG211. A) Representative competition assay using an effective N sucDf:AMG 211 ratio of 2:1. Curve fit with 95% confidence interval is visualized. B) Immunoreactivity towards CEA of different ratios N sucDf:AMG 211. Data are mean {plus minus} SD. Figure S4. Membrane binding and internalization of 89Zr AMG211 after CEA binding on LS174T cells (n = 3). Membrane bound and internalized 89Zr-AMG211 are expressed as percentage of initial cell associated activity. Data are mean {plus minus} SD. Figure S5. Expression of CEA on LS174T, BT474 and HL 60 cell lines (n = 3). Membrane expression is expressed as percentage of LS174T signal. Data are mean {plus minus} SD Table S1. GMP manufacturing of N sucDf AMG211 and 89Zr AMG211. Release criteria are fulfilled for batch 1, 2, and 3. In addition stability are shown for N sucDf AMG211 stored at 80{degree sign}C for 6 months, all quality criteria are still met.

Supplementary Material and Methods / Legends of Supplemental Figures

Purpose:Biodistribution of bispecific antibodies in patients is largely unknown. We therefore performed a feasibility study in 9 patients with advanced gastrointestinal adenocarcinomas to explore AMG 211 biodistribution (also known as MEDI-565), an approximately 55 kDa bispecific T-cell engager (BiTE®) directed against carcinoembryonic antigen (CEA) on tumor cells and cluster of differentiation 3 (CD3) on T-cells.Experimental Design:89Zr-labeled AMG 211 as tracer was administered alone or with cold AMG 211, for PET imaging before and/or during AMG 211 treatment.Results:Before AMG 211 treatment, the optimal imaging dose was 200-μg 89Zr-AMG 211 + 1,800-μg cold AMG 211. At 3 hours, the highest blood pool standardized uptake value (SUV)mean was 4.0, and tracer serum half-life was 3.3 hours. CD3-mediated uptake was clearly observed in CD3-rich lymphoid tissues including spleen and bone marrow (SUVmean 3.2 and 1.8, respectively), and the SUVmean decreased more slowly than in other healthy tissues. 89Zr-AMG 211 remained intact in plasma and was excreted predominantly via the kidneys in degraded forms. Of 43 visible tumor lesions, 37 were PET quantifiable, with a SUVmax of 4.0 [interquartile range (IQR) 2.7–4.4] at 3 hours using the optimal imaging dose. The tracer uptake differed between tumor lesions 5-fold within and 9-fold between patients. During AMG 211 treatment, tracer was present in the blood pool, whereas tumor lesions were not visualized, possibly reflecting target saturation.Conclusions:This first-in-human study shows high, specific 89Zr-AMG 211 accumulation in CD3-rich lymphoid tissues, as well as a clear, inter- and intraindividual heterogeneous tumor uptake.

Multiplex pathology imaging of white-light microscopy, bevacizumab-IRDye800CW fluorescence imaging (green pseudocolor), VEGF-A (red pseudocolor), collagen (red pseudocolor) and CD34 (blue pseudocolor) in breast cancer, including overlay images is shown in a stepwise manner for reasons of clarity.

Purpose: Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is clinically evaluated as novel anticancer drug. rhTRAIL-DR5, a rhTRAIL variant that specifically binds to DR5 receptor, has recently been developed. We investigated whether rhTRAIL-DR5 is more efficient than rhTRAIL in combination with cisplatin in DR5-expressing human A2780 ovarian cancer cells.Design: Effect of cisplatin alone or in combination with rhTRAIL or rhTRAIL-DR5 on DR5 surface expression, apoptosis, and cell survival of A2780 was measured. Biodistribution analysis was done in mice with 125I-rhTRAIL administered intravenously versus intraperitoneally. Antitumor efficacy of rhTRAIL-DR5 versus rhTRAIL was determined in an intraperitoneally growing bioluminescent A2780 xenograft model.Results: Cisplatin strongly enhanced DR5 surface expression. Both rhTRAIL and rhTRAIL-DR5 in combination with cisplatin induced high levels of caspase-3 activation, apoptosis, and cell kill, with rhTRAIL-DR5 being most potent. Intraperitoneal administration of 125I-rhTRAIL resulted in a 1.7-fold higher area under the curve in serum, increased tumor exposure, and more caspase-3 activation in the tumor than intravenous administration. Intraperitoneal administration of rhTRAIL-DR5 delayed A2780 tumor progression, reflected in a mean light reduction of 68.3% (P = 0.015), whereas rhTRAIL or rhTRAIL-DR5 plus cisplatin resulted in 85% (P = 0.003) and 97% (P = 0.002) reduction compared with A2780 tumor progression in vehicle-treated animals. Combination of rhTRAIL-DR5 with cisplatin was more effective than cisplatin alone (P = 0.027).Conclusion: rhTRAIL-DR5 was superior over rhTRAIL in vitro and in vivo against DR5-expressing ovarian cancer also in combination with cisplatin. Intraperitoneal administration of rhTRAIL-DR5 warrants further exploration in ovarian cancer.

Intraoperative and Ex Vivo Optical Imaging of Axillary Lymph Nodes.

Supplementary figure legend

Purpose:Probody therapeutic CX-072 is a protease-activatable antibody that is cross-reactive with murine and human programmed death-ligand 1 (PD-L1). CX-072 can be activated in vivo by proteases present in the tumor microenvironment, thereby potentially reducing peripheral, anti–PD-L1-mediated toxicities. To study its targeting of PD-L1–expressing tissues, we radiolabeled CX-072 with the PET isotope zirconium-89 (89Zr).Experimental Design:89Zr-labeled CX-072, nonspecific Probody control molecule (PbCtrl) and CX-072 parental antibody (CX-075) were injected in BALB/c nude mice bearing human MDA-MB-231 tumors or C57BL/6J mice bearing syngeneic MC38 tumors. Mice underwent serial PET imaging 1, 3, and 6 days after intravenous injection (pi), followed by ex vivo biodistribution. Intratumoral 89Zr-CX-072 distribution was studied by autoradiography on tumor tissue sections, which were subsequently stained for PD-L1 by IHC. Activated CX-072 species in tissue lysates were detected by Western capillary electrophoresis.Results:PET imaging revealed 89Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with 89Zr-PbCtrl. Tumor tissue autoradiography showed high 89Zr-CX-072 uptake in high PD-L1–expressing regions. Activated CX-072 species were detected in these tumors, with 5.3-fold lower levels found in the spleen. Furthermore, 89Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with 89Zr-CX-075, which lacks the Probody design.Conclusions:89Zr-CX-072 accumulates specifically in PD-L1–expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of 89Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491).

Purpose: We evaluated biodistribution and tumor targeting of 89Zr-lumretuzumab before and during treatment with lumretuzumab, a human epidermal growth factor receptor 3 (HER3)–targeting monoclonal antibody.Experimental Design: Twenty patients with histologically confirmed HER3-expressing tumors received 89Zr-lumretuzumab and underwent positron emission tomography (PET). In part A, 89Zr-lumretuzumab was given with additional, escalating doses of unlabeled lumretuzumab, and scans were performed 2, 4, and 7 days after injection to determine optimal imaging conditions. In part B, patients were scanned following tracer injection before (baseline) and after a pharmacodynamic (PD)-active lumretuzumab dose for saturation analysis. HER3 expression was determined immunohistochemically in skin biopsies. Tracer uptake was calculated as standardized uptake value (SUV).Results: Optimal PET conditions were found to be 4 and 7 days after administration of 89Zr-lumretuzumab with 100-mg unlabeled lumretuzumab. At baseline using 100-mg unlabeled lumretuzumab, the tumor SUVmax was 3.4 (±1.9) at 4 days after injection. SUVmean values for normal blood, liver, lung, and brain tissues were 4.9, 6.4, 0.9 and 0.2, respectively. Saturation analysis (n = 7) showed that 4 days after lumretuzumab administration, tumor uptake decreased by 11.9% (±8.2), 10.0% (±16.5), and 24.6% (±20.9) at PD-active doses of 400, 800, and 1,600 mg, respectively, when compared with baseline. Membranous HER3 was completely downregulated in paired skin biopsies already at and above 400-mg lumretuzumab.Conclusions: PET imaging showed biodistribution and tumor-specific 89Zr-lumretuzumab uptake. Although, PD-active lumretuzumab doses decreased 89Zr-lumretuzumab uptake, there was no clear evidence of tumor saturation by PET imaging as the tumor SUV did not plateau with increasing doses. Clin Cancer Res; 23(20); 6128–37. ©2017 AACR.

Microscopic Biodistribution of Bevacizumab-IRDye800CW

Co-localization of Hematoxylin / Eosin (H/E), VEGF-A, and Bevacizumab-IRDye800CW in Tumor-negative Lymph Nodes.

Purpose: Mesothelin (MSLN) is frequently overexpressed in pancreatic and ovarian cancers, making it a potential drug target. We performed an 89Zr-PET imaging study with MMOT0530A, a MSLN antibody, in conjunction with a phase I study with the antibody–drug conjugate DMOT4039A, containing MMOT0530A bound to MMAE. The aim was to study antibody tumor uptake, whole-body distribution, and relation between uptake, response to treatment, and MSLN expression.Experimental Design: Before DMOT4039A treatment, patients received 37 MBq 89Zr-MMOT0530A followed by PET/CT imaging 2, 4, and 7 days postinjection. Tracer uptake was expressed as standardized uptake value (SUV). MSLN expression was determined with immunohistochemistry (IHC) on archival tumor tissue.Results: Eleven patients were included, 7 with pancreatic and 4 with ovarian cancer. IHC MSLN expression varied from absent to strong. Suitable tracer antibody dose was 10 mg MMOT0530A and optimal imaging time was 4 and 7 days postinjection. Tumor tracer uptake occurred in 37 lesions with mean SUVmax of 13.1 (±7.5) on PET 4 days postinjection, with 11.5 (±7.5) in (N = 17) pancreatic and 14.5 (±8.7) in (N = 20) ovarian cancer lesions. Within patients, a mean 2.4-fold (±1.10) difference in uptake between tumor lesions existed. Uptake in blood, liver, kidneys, spleen, and intestine reflected normal antibody distribution. Tracer tumor uptake was correlated to IHC. Best response to DMOT4039A was partial response in one patient.Conclusions: With 89Zr-MMOT0530A-PET, pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized. This technique can potentially guide individualized antibody-based treatment. Clin Cancer Res; 22(7); 1642–52. ©2015 AACR.

Supplemental Figures and Tables

Purpose: HSP90 chaperones have key client proteins that are involved in all hallmarks of breast cancer growth and progression. The primary aim of this clinical trial was to evaluate the feasibility of using 89Zr-trastuzumab PET (for HER2-positive breast cancer) or 89Zr-bevacizumab PET [for estrogen receptor (ER)–positive breast cancer] to determine in vivo degradation of client proteins caused by the novel HSP90 inhibitor NVP-AUY922.Experimental Design: Of note, 70 mg/m2 NVP-AUY922 was administered intravenously in a weekly schedule to patients with advanced HER2 or ER-positive breast cancer. Biomarker analysis consisted of serial PET imaging with 2[18F]fluoro-2-deoxy-D-glucose (FDG), 89Zr-trastuzumab, or 89Zr-bevacizumab. Response evaluation was performed according to RECIST1.0. FDG, 89Zr-trastuzumab, and 89Zr-bevacizumab distributions were scored visually and quantitatively by calculating the maximum standardized uptake values (SUVmax). In blood samples, serial HSP70 levels, extracellular form of HER2 (HER2-ECD), and pharmacokinetic and pharmacodynamic parameters were measured.Results: Sixteen patients (ten HER2-positive and six ER-positive tumors) were included. One partial response was observed; seven patients showed stable disease. SUVmax change in individual tumor lesions on baseline versus 3 weeks 89Zr-trastuzumab PET was heterogeneous and related to size change on CT after 8 weeks treatment (r2 = 0.69; P = 0.006). Tumor response on 89Zr-bevacizumab PET and FDG-PET was not correlated with CT response.Conclusions: NVP-AUY922 showed proof-of-concept clinical response in HER2-amplified metastatic breast cancer. Early change on 89Zr-trastuzumab PET was positively associated with change in size of individual lesions assessed by CT. Clin Cancer Res; 20(15); 3945–54. ©2014 AACR.

Supplementary Data from Enhanced Antitumor Efficacy of a DR5-Specific TRAIL Variant over Recombinant Human TRAIL in a Bioluminescent Ovarian Cancer Xenograft Model

Purpose:CX-072, a PD-L1–targeting Probody therapeutic, is engineered to be activated by tumor proteases that remove a masking peptide. To study effects on biodistribution and pharmacokinetics, we performed 89Zr-CX-072 positron emission tomography (PET) imaging.Experimental Design:Patients received ∼1 mg, 37 MBq 89Zr-CX-072 plus 0, 4, or 9 mg unlabeled CX-072 and PET scans at days 2, 4, and 7. After that, treatment comprised 10 mg/kg CX-072 q2 weeks (n = 7) + 3 mg/kg ipilimumab q3w 4× (n = 1). Normal organ tracer uptake was expressed as standardized uptake value (SUV)mean and tumor uptake as SUVmax. PD-L1 expression was measured immunohistochemically in archival tumor tissue.Results:Three of the eight patients included received 10-mg protein dose resulting in a blood pool mean SUVmean ± SD of 4.27 ± 0.45 on day 4, indicating sufficient available tracer. Tumor uptake was highest at day 7, with a geometric mean SUVmax 5.89 (n = 113) and present in all patients. The median follow-up was 12 weeks (4–76+). One patient experienced stable disease and two patients a partial response. PD-L1 tumor expression was 90% in one patient and ≤1% in the other patients. Mean SUVmean ± SD day 4 at 10 mg in the spleen was 8.56 ± 1.04, bone marrow 2.21 ± 0.46, and liver 4.97 ± 0.97. Four patients out of seven showed uptake in normal lymph nodes and Waldeyer's ring. The tracer was intact in the serum or plasma.Conclusions:89Zr-CX-072 showed tumor uptake, even in lesions with ≤1% PD-L1 expression, and modest uptake in normal lymphoid organs, with no unexpected uptake in other healthy tissues.

Representative Fluorescence Flatbed Scans of Paraffin Blocks of all Patients (N=20) and Macroscopic Segmentation of Fluorescence Tissue Localization and Target-to-Background Ratios (TBR).

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of 89Zr-trastuzumab (SKOV-3 and OE19), 89Zr-bevacizumab (SKOV-3), or 89Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment. Cancer Res; 73(11); 3347–55. ©2013 AACR.

Supplementary Figure Legends 1-7 from VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment