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Figure S3: Ex-vivo biodistribution study of PD-1 i.p and PBS i.p groups (n{greater than or equal to}5) on Day 8 post anti PD-1 treatment: the quantification of TDLN was expressed as %ID, other organs were expressed as %ID/g. All values represent the mean {plus minus} SD unless otherwise specified. Unpaired 2-tailed student's t-test were used for analysis. (*: P

Supplementary movie 1. Boundary selection software

Supplementary Figures - PDF file 110K, Supplementary Figure 1. Dose titration curves of ficlatuzumab. The numbers in parenthesis are the median survival in days. Supplementary Figure 2. combination therapy of ficlatuzumab and temozolomide studied in an intracranial U87 MG xenograft mouse model. The numbers in parenthesis are the median survival in days. Supplementary Figure 3. Comparison of U87 MG parental and U87 MG-Luc2 cells showing similarity in in vitro (a) and in vivo (b) characteristics. Supplementary Figure 4. Change in weight (mean � standard deviation) over the course of the experiment in the control, low-dose, and high-dose groups. Results shown are normalized to the baseline value. The p-values shown are the results of the ANOVA for each group. In this case, there were no significant changes in weight

Supplemental Materials and Methods. A detailed description of the radiosynthesis of each radiotracer, and detailed PET and SPECT imaging protocols.

Detailed overview of 510(k) cleared imaging devices by the FDA.

Supplemental Fig. S2 Calculation of Total Lesion Immune response (TLIR). When 3D volume of interest (VOI) was placed to include a targeted lesion (green circle), Osirix MD software automatically calculates the volume above 42% of the maximum uptake within the VOI (purple area). The threshold 42% was applied following the majority of previous clinical PET studies. TLIR was defined as mean tracer uptake within the targeted VOI multiplied by volume above 42% of maximum uptake.

Movie S1

Purpose: An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of 18F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3–triggered nanoaggregation.Experimental Design: Here, we compared the preclinical utility of 18F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, 18F-FDG, 99mTc-Annexin V, and 18F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo.Results: In drug-treated lymphoma cells, 18F-FDG, 99mTc-Annexin V, and 18F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R2 > 0.8; P < 0.001), with no correlation measured for 18F-ML-10 (R2 = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of 99mTc-Annexin V and 18F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although 99mTc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either 18F-FDG or 18F-ML-10.Conclusions: We have demonstrated here that 18F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with 18F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility. Clin Cancer Res; 21(17); 3896–905. ©2015 AACR.

Figure S1 shows mass spectometry results of the targeting agent (A), and control peptide (B). It also shows photostability of agent. Figure S2 shows characterisation of the different cell lines used. Figure S3 shows the difference in fluorescence between PDAC and peritumoral inflammation Figure S4 shows results of the preliminary toxicity study in mice

Supplemental Video M3. 18FML-10 uptake in naive tumors

PDF file - 1.3MB, Figures, Tables, Methods.

Supplementary Figure 3. MALDI-MS of TIGITAb and DFO-TIGITAb demonstrating successful bioconjugation.

Table S1: Characterization of 89Zr-DFO-ICOS mAb, n=4

Day4_GVHD

List of legend for supplementary files. Description list for supplementary files

Purpose: (4S)-4-(3-[18F]fluoropropyl)-l-glutamate (BAY 94-9392, alias [18F]FSPG) is a new tracer to image xC− transporter activity with positron emission tomography (PET). We aimed to explore the tumor detection rate of [18F]FSPG in patients relative to 2-[18F]fluoro-2-deoxyglucose ([18F]FDG). The correlation of [18F]FSPG uptake with immunohistochemical expression of xC− transporter and CD44, which stabilizes the xCT subunit of system xC−, was also analyzed.Experimental Design: Patients with non–small cell lung cancer (NSCLC, n = 10) or breast cancer (n = 5) who had a positive [18F]FDG uptake were included in this exploratory study. PET images were acquired following injection of approximately 300 MBq [18F]FSPG. Immunohistochemistry was done using xCT- and CD44-specific antibody.Results: [18F]FSPG PET showed high uptake in the kidney and pancreas with rapid blood clearance. [18F]FSPG identified all 10 NSCLC and three of the five breast cancer lesions that were confirmed by pathology. [18F]FSPG detected 59 of 67 (88%) [18F]FDG lesions in NSCLC, and 30 of 73 (41%) in breast cancer. Seven lesions were additionally detected only on [18F]FSPG in NSCLC. The tumor-to-blood pool standardized uptake value (SUV) ratio was not significantly different from that of [18F]FDG in NSCLC; however, in breast cancer, it was significantly lower (P < 0.05). The maximum SUV of [18F]FSPG correlated significantly with the intensity of immunohistochemical staining of xC− transporter and CD44 (P < 0.01).Conclusions: [18F]FSPG seems to be a promising tracer with a relatively high cancer detection rate in patients with NSCLC. [18F]FSPG PET may assess xC− transporter activity in patients with cancer. Clin Cancer Res; 18(19); 5427–37. ©2012 AACR.

Supplementary information

Purpose:To design and evaluate a small engineered protein binder targeting human programmed death-1 ligand (hPD-L1) in vivo for PET imaging in four mouse tumor models, and in situ in human cancer specimens.Experimental Design: The hPD-L1 protein binder, FN3hPD-L1, was engineered using a 12-kDa human fibronectin type-3 domain (FN3) scaffold. The binder's affinity was assayed in CT26 mouse colon carcinoma cells stably expressing hPD-L1 (CT26/hPD-L1). 64Cu-FN3hPD-L1 was assayed for purity, specific activity, and immunoreactivity. Four groups of NSG mice (n = 3–5/group) were imaged with 64Cu-FN3hPD-L1 PET imaging (1–24 hours postinjection of 3.7 MBq/7 μg of Do-FN3 in 200 μL PBS): Nod SCID Gamma (NSG) mice bearing (i) syngeneic CT26/hPD-L1tumors, (iihPD-L1 binder, (iiihPD-L1 binder staining was evaluated against validated hPD-L1 antibodies by immunostaining in human cancer specimens.Results:FN3hPD-L1 bound hPD-L1 with 1.4 ± 0.3 nmol/L affinity in CT26/hPD-L1 cells. 64Cu-FN3hPD-L1 radiotracer showed >70% yield and >95% purity. 64Cu-FN3hPD-L1 PET imaging of mice bearing CT26/hPD-L1 tumors showed tumor-to-muscle ratios of 5.6 ± 0.9 and 13.1 ± 2.3 at 1 and 4 hours postinjection, respectively. The FN3hPD-L1 binder detected hPD-L1 expression in human tissues with known hPD-L1 expression status based on two validated antibodies.Conclusions:The 64Cu-FN3hPD-L1 radiotracer represents a novel, small, and high-affinity binder for imaging hPD-L1 in tumors. Our data support further exploration and clinical translation of this binder for noninvasive identification of cancer patients who may respond to immune checkpoint blockade therapies.

Purpose: To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbubble–enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC).Experimental Design: Thy1-scFv were engineered using yeast-surface-display techniques. Binding to soluble human and murine Thy1 and to Thy1-expressing cells was assessed by flow cytometry. Thy1-scFv was then attached to gas-filled microbubbles to create MBThy1-scFv. Thy1 binding of MBThy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MBscFv-scrambled and MBNon-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice in vivo.Results: Thy1-scFv had a KD of 3.4 ± 0.36 nmol/L for human and 9.2 ± 1.7 nmol/L for murine Thy1 and showed binding to both soluble and cellularly expressed Thy1. MBThy1-scFv was attached to Thy1 with high affinity compared with negative control microbubbles (P < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (P < 0.01) higher binding of MBThy1-scFv (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MBscFv-scrambled (0.57 ± 0.53) and MBNon-targeted (0.43 ± 0.53). In vivo ultrasound molecular imaging using MBThy1-scFv demonstrated significantly higher signal (P < 0.01) in both orthotopic (5.32 ± 1.59 a.u.) and transgenic PDAC (5.68 ± 2.5 a.u.) mice compared with chronic pancreatitis (0.84 ± 0.6 a.u.) and normal pancreas (0.67 ± 0.71 a.u.). Ex vivo immunofluorescence confirmed significantly (P < 0.01) increased Thy1 expression in PDAC compared with chronic pancreatitis and normal pancreas tissue.Conclusions: A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound. Clin Cancer Res; 24(7); 1574–85. ©2018 AACR.

PDF file, 76K, includes movie legends.

AVI, 180K, Maximum intensity projection of 18FFSPG in a lung cancer patient (Patient 2).

Figure S6: The bar graphs above show the FACS analysis of ICOS expression (%) at Day 2 and Day 8 measured on CD4 and CD8 T cells from TDLN. On Day 2, anti PD-1 treated mice showed upregulation of ICOS in CD4 and CD8 T cells, compared to that of PBS group (~ 2-fold in CD4 T cells and 2.5-fold in CD8 T cells). On Day 8, ICOS levels on both CD4+ and CD8+ T cells decreased in the anti PD-1 treatment group, relative to that of Day 2, and PBS group remained similar with Day 2. (*: P

Supplementary Figure Legend - PDF file 60K, Supplementary figure legend

Figure S5 from A Novel Engineered Small Protein for Positron Emission Tomography Imaging of Human Programmed Death Ligand-1: Validation in Mouse Models and Human Cancer Tissues

Supplementary Methods

Figure S4: Correlation of Tumor and TDLN PET ROI with fold-change in tumor volume (Day8/Day0) in mice treated with anti PD-1 antibody i.p and PBS i.p.

Figure S5: Quantification of the relative proportion of Foxp3+ compared to Foxp3- CD4 T cells in the lymph node of mice treated with anti PD-1 antibody. (****: P

Figure S1: Stability of 89Zr-DFO-ICOS mAb in PBS and serum

AVI, 161K, Maximum intensity projection of 18FFSPG in a breast cancer patient (Patient 13).

Purpose: Ficlatuzumab is a novel therapeutic agent targeting the hepatocyte growth factor (HGF)/c-MET pathway. We summarize extensive preclinical work using this agent in a mouse brain orthotopic model of glioblastoma.Experimental Design: Sequential experiments were done using eight- to nine-week-old nude mice injected with 3 × 105 U87 MG (glioblastoma) cells into the brain. Evaluation of ficlatuzumab dose response for this brain tumor model and comparison of its response to ficlatuzumab and to temozolamide were conducted first. Subsequently, various small-animal imaging modalities, including bioluminescence imaging (BLI), positron emission tomography (PET), and MRI, were used with a U87 MG-Luc 2 stable cell line, with and without the use of ficlatuzumab, to evaluate the ability to noninvasively assess tumor growth and response to therapy. ANOVA was conducted to evaluate for significant differences in the response.Results: There was a survival benefit with ficlatuzumab alone or in combination with temozolamide. BLI was more sensitive than PET in detecting tumor cells. Fluoro-D-thymidine (FLT) PET provided a better signal-to-background ratio than 2[18F]fluoro-2-deoxy-d-glucose (FDG) PET. In addition, both BLI and FLT PET showed significant changes over time in the control group as well as with response to therapy. MRI does not disclose any time-dependent change. Also, the MRI results showed a temporal delay in comparison to the BLI and FLT PET findings, showing similar results one drug cycle later.Conclusions: Targeting the HGF/c-MET pathway with the novel agent ficlatuzumab appears promising for the treatment of glioblastoma. Various clinically applicable imaging modalities including FLT, PET, and MRI provide reliable ways of assessing tumor growth and response to therapy. Given the clinical applicability of these findings, future studies on patients with glioblastoma may be appropriate. Clin Cancer Res; 19(20); 5711–21. ©2013 AACR.

Supplemental Figures & Legends

PDF file, 358K, S1: System xC� and CD44 in glutathione biosynthesis and the cysteine-cystine redox cycle; S2: 18FFSPG distribution in normal organs and cancer; S3: Correlation between 18FFDG and 18FFSPG; S4: 18FFDG and 18FFSPG PET in a breast cancer patient; S5: Correlation analysis between the staining intensity of xC� transporter and CD44 expression.

PDF file, 72K, S1: Clinical safety laboratory parameters; S2: Tissue distribution of 18FFSPG.

Purpose: To evaluate the utility of targeted photoacoustic imaging (PAI) in providing molecular information to complement intrinsic functional and anatomical details of the vasculature within prostate lesion.Experimental Design: We developed a PAI agent, AA3G-740, that targets gastrin-releasing peptide receptor (GRPR), found to be highly overexpressed in prostate cancer. The binding specificity of the agent was evaluated in human prostate cancer cell lines, PC3 and LNCaP, and antagonist properties determined by cell internalization and intracellular calcium mobilization studies. The imaging sensitivity was assessed for the agent itself and for the PC3 cells labeled with agent. The in vivo stability of the agent was determined in human plasma and in the blood of living mice. The in vivo binding of the agent was evaluated in PC3 prostate tumor models in mice, and was validated ex vivo by optical imaging.Results: AA3G-740 demonstrated strong and specific binding to GRPR. The sensitivity of detection in vitro indicated suitability of the agent to image very small lesions. In mice, the agent was able to bind to GRPR even in poorly vascularized tumors leading to nearly 2-fold difference in photoacoustic signal relative to the control agent.Conclusions: The ability to image both vasculature and molecular profile outside the blood vessels gives molecular PAI a unique advantage over currently used imaging techniques. The imaging method presented here can find application both in diagnosis and in image-guided biopsy. Clin Cancer Res; 20(14); 3721–9. ©2014 AACR.

Considerable advances in cancer-specific optical imaging have improved the precision of tumor resection. In comparison to traditional imaging modalities, this technology is unique in its ability to provide real-time feedback to the operating surgeon. Given the significant clinical implications of optical imaging, there is an urgent need to standardize surgical navigation tools and contrast agents to facilitate swift regulatory approval. Because fluorescence-enhanced surgery requires a combination of both device and drug, each may be developed in conjunction, or separately, which are important considerations in the approval process. This report is the result of a one-day meeting held on May 4, 2016 with officials from the National Cancer Institute, the FDA, members of the American Society of Image-Guided Surgery, and members of the World Molecular Imaging Society, which discussed consensus methods for FDA-directed human testing and approval of investigational optical imaging devices as well as contrast agents for surgical applications. The goal of this workshop was to discuss FDA approval requirements and the expectations for approval of these novel drugs and devices, packaged separately or in combination, within the context of optical surgical navigation. In addition, the workshop acted to provide clarity to the research community on data collection and trial design. Reported here are the specific discussion items and recommendations from this critical and timely meeting. Cancer Res; 77(9); 2197–206. ©2017 AACR.

Purpose:Therapeutic checkpoint inhibitors on tumor-infiltrating lymphocytes (TIL) are being increasingly utilized in the clinic. The T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T and natural killer cells. The TIGIT signaling pathway is an alternative target for checkpoint blockade to current PD-1/CTLA-4 strategies. Elevated TIGIT expression in the tumor microenvironment correlates with better therapeutic responses to anti-TIGIT therapies in preclinical models. Therefore, quantifying TIGIT expression in tumors is necessary for determining whether a patient may respond to anti-TIGIT therapy. PET imaging of TIGIT expression on TILs can therefore aid diagnosis and in monitoring therapeutic responses.Experimental Design:Antibody-based TIGIT imaging radiotracers were developed with the PET radionuclides copper-64 (64Cu) and zirconium-89 (89Zr). In vitro characterization of the imaging probes was followed by in vivo evaluation in both xenografts and syngeneic tumor models in mouse.Results:Two anti-TIGIT probes were developed and exhibited immunoreactivity of >72%, serum stability of >95%, and specificity for TIGIT with both mouse TIGIT-expressing HeLa cells and ex vivo–activated primary splenocytes. In vivo, the 89Zr-labeled probe demonstrated superior contrast than the 64Cu probe due to 89Zr's longer half-life matching the TIGIT antibody's pharmacokinetics. The 89Zr probe was used to quantify TIGIT expression on TILs in B16 melanoma in immunocompetent mice and confirmed by ex vivo flow cytometry.Conclusions:This study develops and validates novel TIGIT-specific 64Cu and 89Zr PET probes for quantifying TIGIT expression on TILs for diagnosis of patient selection for anti-TIGIT therapies.

Purpose: Intraoperative near-infrared fluorescence (NIRF) imaging could help stratification for the proper primary treatment for patients with pancreatic ductal adenocarcinoma (PDAC), and achieve complete resection, as it allows visualization of cancer in real time. Integrin αvβ6, a target specific for PDAC, is present in >90% of patients, and is able to differentiate between pancreatitis and PDAC. A clinically translatable αvβ6-targeting NIRF agent was developed, based on a previously developed cysteine knottin peptide for PET imaging, R01-MG, and validated in preclinical mouse models.Experimental Design: The applicability of the agent was tested for cell and tissue binding characteristics using cell-based plate assays, subcutaneous, and orthotopic pancreatic models, and a transgenic mouse model of PDAC development (Pdx1-Cretg/+;KRasLSL G12D/+;Ink4a/Arf−/−). IRDye800CW was conjugated to R01-MG in a 1:1 ratio. R01-MG-IRDye800, was compared with a control peptide and IRDye800 alone.Results: In subcutaneous tumor models, a significantly higher tumor-to-background ratio (TBR) was seen in BxPC-3 tumors (2.5 ± 0.1) compared with MiaPaCa-2 (1.2 ± 0.1; P < 0.001), and to the control peptide (1.6 ± 0.4; P < 0.005). In an orthotopic tumor model, tumor-specific uptake of R01-MG-IRDye800 was shown compared with IRDye800 alone (TBR 2.7 vs. 0.86). The fluorescent signal in tumors of transgenic mice was significantly higher, TBR of 3.6 ± 0.94, compared with the normal pancreas of wild-type controls, TBR of 1.0 ± 0.17 (P < 0.001).Conclusions: R01-MG-IRDye800 shows specific targeting to αvβ6, and holds promise as a diagnostic and therapeutic tool to recognize PDAC for fluorescence-guided surgery. This agent can help improve the stratification of patients for a potentially curative, margin-negative resection. Clin Cancer Res; 24(7); 1667–76. ©2018 AACR.

Supplemental Figure legends

Purpose: To determine if magnetotactic bacteria can target tumors in mice and provide positive contrast for visualization using magnetic resonance imaging.Experimental Design: The ability of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1 (referred to from here as AMB-1), to confer positive magnetic resonance imaging contrast was determined in vitro and in vivo. For the latter studies, AMB-1 were injected either i.t. or i.v. Bacterial growth conditions were manipulated to produce small (∼25-nm diameter) magnetite particles, which were observed using transmission electron microscopy. Tumor targeting was confirmed using 64Cu-labeled bacteria and positron emission tomography and by determination of viable cell counts recovered from different organs and the tumor.Results: We show that AMB-1 bacteria with small magnetite particles generate T1-weighted positive contrast, enhancing in vivo visualization by magnetic resonance imaging. Following i.v. injection of 64Cu-labeled AMB-1, positron emission tomography imaging revealed increasing colonization of tumors and decreasing infection of organs after 4 hours. Viable cell counts showed that, by day 6, the bacteria had colonized tumors but were cleared completely from other organs. Magnetic resonance imaging showed a 1.22-fold (P = 0.003) increased positive contrast in tumors on day 2 and a 1.39-fold increase (P = 0.0007) on day 6.Conclusion: Magnetotactic bacteria can produce positive magnetic resonance imaging contrast and colonize mouse tumor xenografts, providing a potential tool for improved magnetic resonance imaging visualization in preclinical and translational studies to track cancer. (Clin Cancer Res 2009;15(16):5170–7)

Purpose: We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity.Experimental Design: Four PET tracers [18F-FDG, 18F-Fpp(RGD)2, 18F-FLT, and 18F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes.Results: Significant changes in uptake of 18F-FAZA, 18F-FLT, and 18F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with 18F-FAZA being the most prominent. 18F-FDG uptake was unaffected. 18F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. 18F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting 18F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced 18F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed.Conclusions: Our studies suggest suitability of 18F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243). Clin Cancer Res; 21(2); 335–46. ©2014 AACR.

Purpose:Immunotherapy is a promising approach for many oncological malignancies, including glioblastoma, however, there are currently no available tools or biomarkers to accurately assess whole-body immune responses in patients with glioblastoma treated with immunotherapy. Here, the utility of OX40, a costimulatory molecule mainly expressed on activated effector T cells known to play an important role in eliminating cancer cells, was evaluated as a PET imaging biomarker to quantify and track response to immunotherapy.Experimental Design:A subcutaneous vaccination approach of CpG oligodeoxynucleotide, OX40 mAb, and tumor lysate at a remote site in a murine orthotopic glioma model was developed to induce activation of T cells distantly while monitoring their distribution in stimulated lymphoid organs with respect to observed therapeutic effects. To detect OX40-positive T cells, we utilized our in-house–developed 89Zr-DFO-OX40 mAb and in vivo PET/CT imaging.Results:ImmunoPET with 89Zr-DFO-OX40 mAb revealed strong OX40-positive responses with high specificity, not only in the nearest lymph node from vaccinated area (mean, 20.8%ID/cc) but also in the spleen (16.7%ID/cc) and the tumor draining lymph node (11.4%ID/cc). When the tumor was small (6 p/sec/cm2/sr in bioluminescence imaging), a high number of responders and percentage shrinkage in tumor signal was indicated after only a single cycle of vaccination.Conclusions:The results highlight the promise of clinically translating cancer vaccination as a potential glioma therapy, as well as the benefits of monitoring efficacy of these treatments using immunoPET imaging of T-cell activation.

Thyroid cancer is one of the most common cancers, with a global increase in incidence rate for both genders. Ultrasound-guided fine-needle aspiration is the current gold standard to diagnose thyroid cancers, but the results are inaccurate, leading to repeated biopsies and unnecessary surgeries. To reduce the number of unnecessary biopsies, we explored the use of multiparametric photoacoustic (PA) analysis in combination with the American Thyroid Association (ATA) Guideline (ATAP). In this study, we performed in vivo multispectral PA imaging on thyroid nodules from 52 patients, comprising 23 papillary thyroid cancer (PTC) and 29 benign cases. From the multispectral PA data, we calculated hemoglobin oxygen saturation level in the nodule area, then classified the PTC and benign nodules with multiparametric analysis. Statistical analyses showed that this multiparametric analysis of multispectral PA responses could classify PTC nodules. Combining the photoacoustically indicated probability of PTC and the ATAP led to a new scoring method that achieved a sensitivity of 83% and a specificity of 93%. This study is the first multiparametric analysis of multispectral PA data of thyroid nodules with statistical significance. As a proof of concept, the results show that the proposed new ATAP scoring can help physicians examine thyroid nodules for fine-needle aspiration biopsy, thus reducing unnecessary biopsies.Significance:This report highlights a novel photoacoustic scoring method for risk stratification of thyroid nodules, where malignancy of the nodules can be diagnosed with 83% sensitivity and 93% specificity.

Supplemental Figures S1-4 and Tables 1-2. Supplemental Figure S1. Mechanism of 18F-C-SNAT activation, cyclization and intracellular trapping; Supplemental Figure S2. Flow cytometric analysis of cell death in EL-4 cell mixtures; Supplemental Figure S3. Radiotracer uptake and retention in carboplatin- and vehicle-treated PC9 and A549 non small cell lung cancer cells; Supplemental Figure S4. SPECT imaging of 99mTc-Annexin V uptake in vivo; Supplemental Table 1. Ex vivo biodistribution data for selected tissues in naive and drug-treated mice 90 min after injection; Supplemental Table 2. Radiotracer tumor-to-tissue ratios in naive and drug-treated EL-4 xenografts 90 min after injection.

Purpose: To determine whether optical imaging can be used for in vivo therapy response monitoring as an alternative to radionuclide techniques. For this, we evaluated the known Her2 response to 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) treatment, an Hsp90 inhibitor.Experimental Design: After in vitro 17-DMAG treatment response evaluation of MCF7 parental cells and 2 HER2-transfected clones (clone A medium, B high Her2 expression), we established human breast cancer xenografts in nude mice (only parental and clone B) for in vivo evaluation. Mice received 120 mg/kg of 17-DMAG in 4 doses at 12-hour intervals intraperitonially (n = 14) or PBS as carrier control (n = 9). Optical images were obtained both pretreatment (day 0) and posttreatment (day 3, 6, and 9), always 5 hours postinjection of 500 pmol of anti-Her2 Affibody-AlexaFluor680 via tail vein (with preinjection background subtraction). Days 3 and 9 in vivo optical imaging signal was further correlated with ex vivo Her2 levels by Western blot after sacrifice.Results: Her2 expression decreased with 17-DMAG dose in vitro. In vivo optical imaging signal was reduced by 22.5% in clone B (P = 0.003) and by 9% in MCF7 parental tumors (P = 0.23) 3 days after 17-DMAG treatment; optical imaging signal recovered in both tumor types at days 6 to 9. In the carrier group, no signal reduction was observed. Pearson correlation of in vivo optical imaging signal with ex vivo Her2 levels ranged from 0.73 to 0.89.Conclusions: Optical imaging with an affibody can be used to noninvasively monitor changes in Her2 expression in vivo as a response to treatment with an Hsp90 inhibitor, with results similar to response measurements in positron emission tomography imaging studies. Clin Cancer Res; 18(4); 1073–81. ©2012 AACR.

PDF file - 232K

Supporting Information from Optimizing Radiolabeled Engineered Anti-p185HER2 Antibody Fragments for In vivo Imaging

Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB–CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models. Cancer Res; 70(24); 10141–9. ©2010 AACR.

PDF file - 116K, Table S1: Summary of Sensitivity, Specificity, Positive and Negative Predictive Values for Differentiating Benign (Normal, Hyperplasia) from Malignant (DCIS, Invasive Cancer) Pathologies Based on Different Imaging Signal Threshold Levels; Table S2a: Summary of Sensitivity, Specificity, Positive and Negative Predictive Values for Differentiating Benign (Normal, Hyperplasia) from DCIS Based on Different Imaging Signal Threshold Levels; Table S2b: Summary of Sensitivity, Specificity, Positive and Negative Predictive Values for Differentiating Benign (Normal, Hyperplasia) from Invasive Cancer Based on Different Imaging Signal Threshold Levels.

Supplementary Movie 1 from PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model with a Novel 64Cu-DOTA-Knottin Peptide

Due to the high mortality of lung cancer, there is a critical need to develop diagnostic procedures enabling early detection of the disease while at a curable stage. Targeted molecular imaging builds on the positive attributes of positron emission tomography/computed tomography (PET/CT) to allow for a noninvasive detection and characterization of smaller lung nodules, thus increasing the chances of positive treatment outcome. In this study, we investigate the ability to characterize lung tumors that spontaneously arise in a transgenic mouse model. The tumors are first identified with small animal CT followed by characterization with the use of small animal PET with a novel 64Cu–1,4,7,10-tetra-azacylododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)–knottin peptide that targets integrins upregulated during angiogenesis on the tumor associated neovasculature. The imaging results obtained with the knottin peptide are compared with standard 18F-fluorodeoxyglucose (FDG) PET small animal imaging. Lung nodules as small as 3 mm in diameter were successfully identified in the transgenic mice by small animal CT, and both 64Cu-DOTA-knottin 2.5F and FDG were able to differentiate lung nodules from the surrounding tissues. Uptake and retention of the 64Cu-DOTA-knottin 2.5F tracer in the lung tumors combined with a low background in the thorax resulted in a statistically higher tumor to background (normal lung) ratio compared with FDG (6.01 ± 0.61 versus 4.36 ± 0.68; P < 0.05). Ex vivo biodistribution showed 64Cu-DOTA-knottin 2.5F to have a fast renal clearance combined with low nonspecific accumulation in the thorax. Collectively, these results show 64Cu-DOTA-knottin 2.5F to be a promising candidate for clinical translation for earlier detection and improved characterization of lung cancer. Cancer Res; 70(22); 9022–30. ©2010 AACR.