Tumor-specific Signal Research Articles

Abstract LB115: Simultaneous whole-body ultrasound contrast imaging: a novel high throughput preclinical platform for targeted molecular imaging studies

Abstract Ultrasound contrast agent (UCA) based molecular imaging is a rapidly growing field in biomedical research. Ultrasound (US) is a non-radiative imaging modality that is frequently used to investigate various human diseases in the clinic. US molecular imaging is a novel imaging modality that utilizes targeted contrast agents to add molecular information about the imaging target, including vascular biomarkers of cancer. Pre-clinical animal models are commonly used to evaluate and validate targeted contrast agents before extending them for FDA-approval. Key impediments in the preclinical evaluations are the short in vivo half-life of microbubbles (MBs) (less than 30 minutes), limited transducer scanning area, and focusing one target at a time, which all make this strategy low throughput. Moreover, whole-body imaging strategy is not available for US imaging compared to other imaging modalities, such as PET, CT, and MRI. To overcome these imitations, we have developed a multi-mouse US imaging platform using an Automated Breast Volume Scanner (ABVS) system. In addition, we have synthesized a novel breast cancer vascular-specific B7-H3 targeted microbubbles (TMBs) using a microfluidic device. We tested these TMBs in a genetically engineered transgenic mice model [FVB/N-Tg (MMTV-PyMT)634Mul/J] (n=32) that develops breast cancer in the mammary fat pad. The US imaging results of conventional Vevo 2100 system indicated that B7-H3-TMBs could provide target specific contrast signals in the tumor vasculature compared to control MBs. We used this MBs for whole-body imaging using an ABVS system with two mice simultaneously in a stage. We imaged multiple tumors (10 tumors/mouse, 2 mice per scan, n=20 tumors) with a single bolus injection of TMBs per mouse. The results successfully showed tumor specific contrast signals from tumors independent of tumor size. Introduction of this whole-body scanner, and its ability to scan multiple mice simultaneously allows rapid imaging of multiple tumors with 3D quantification of tumor size by administering a single TMB injection enabling without significant contrast loss. Our approach makes this US-MI as high throughput and can be used to test multiple contrast agents for investigating different aspects of biological problems including screening of vascular biomarkers of human diseases. Citation Format: Arutselvan Natarajan, Farbod Tabesh, Dongwoon Hyun, Ramasamy Paulmurugan, Jeremy Dahl. Simultaneous whole-body ultrasound contrast imaging: a novel high throughput preclinical platform for targeted molecular imaging studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB115.

EP01.01-006 Sensitive Detection of Lung Cancer Using a Multiomic Plasma Cell-Free DNA Sequencing Assay

Cancer detection at early stages expands therapeutic options and improves treatment efficacy. Existing cancer screening methods, however, are often invasive and have limited accuracy, subjecting patients to unnecessary follow-up procedures. Detection of plasma circulating tumor DNA (ctDNA) is a reliable method for detecting cancer, but sensitivity can be limited. Methylation signatures in cell-free DNA (cfDNA) can enhance cancer detection by providing cancer-specific epigenetic information. In addition, cfDNA from tumor cells are relatively shorter than cfDNA from normal cells, and this difference can be measured as a tumor-specific signal.

Glioblastoma Associated Natural Killer Cell EVs Generating Tumour-Specific Signatures: Noninvasive GBM Liquid Biopsy with Self-Functionalized Quantum Probes.

Diagnosis of glioblastoma (GBM) poses a recurring struggle due to many factors, including the presence of the blood-brain barrier (BBB) in addition to the significant tumor heterogeneity. Natural killer (NK) cells of the innate immune system are the primary immune surveillance mechanism for GBM and identify GBM tumors without any previous sensitization. The metabolic reprogramming of NK cells during GBM association is expected to be reflected in its extracellular vesicles. Therefore, tracking the activity of NK cell vesicles in circulation (circulating immune vesicles, CIVs) has great potential for accurate GBM diagnosis. However, identification GBM associated CIVs in circulation is immensely challenging as there is no availability of clinically validated GBM-specific circulating biomarkers. Here, we present GBM associated CIV profiling for noninvasive GBM diagnosis. We investigated the feasibility of using the signals derived from GBM associated CIVs as a de novo methodology for GBM diagnosis. An ultrasensitive sensor and a marker-free approach were essential for the detection of rare signals of GBM associated CIVs. For this purpose, we designed GBM ImmunoProfiler platform using scalable ultrafast laser multiphoton ionization mechanism and adopted surface enhanced Raman spectroscopy (SERS) ensuring simultaneous detection of multiple CIV signals to identify GBM. We experimentally demonstrated that GBM associated CIVs carry unique, tumor-specific signals. The features of GBM associated CIVs were explored through machine learning identifying its similarity with GBM patient blood (without cell isolation) using a very small amount of peripheral blood (5 μL) with 96.82% sensitivity and 100% specificity. In addition, we demonstrated that a tumor associated CIV profile can classify between multiple brain cancer types (astrocytoma, oligodendroglioma, and glioblastoma). We also experimentally demonstrated significant variation in the immune checkpoint protein expression (PDL-1 and CTLA-4) between GBM associated CIVs and uninteracted CIVs. Preclinical analysis with serum specimens of GBM patients showed the possibility of using our technology for minimally invasive GBM diagnosis. With clinical validation, our technology has potential to improve GBM diagnostics with a useful, minimally invasive GBM liquid biopsy.

Early detection of cancer using cell-free DNA (cfDNA) size analysis on a multiplexed amplicon-based next-generation sequencing (NGS) platform.

3041 Background: Tumor-derived cfDNA fragments are observed to be shorter in length than normal cfDNA. This size (length) difference can be analyzed as a tumor-specific signal. Whole genome or probe hybridization-based NGS methods can capture cfDNA fragments of native sizes. However, amplicon-based NGS assays are not directly amenable to cfDNA size analysis due to predetermination of amplicon sizes by design. Here we present a method to extract relative distribution of cfDNA fragment lengths from a targeted amplicon-based assay and show its utility in cancer detection. Methods: The LiquidHALLMARK cfDNA assay is an amplicon-based NGS test for the sensitive detection of genomic alterations in 80 genes. Although panel design is optimized for cfDNA with average amplicon length of ̃150 bp, the consecutive tiling design of amplicons for genes with contiguously targeted regions, e.g. BRCA permits the formation of longer amplicons (> 150 bp) from physically subsequent primer pairs, provided longer template cfDNA molecules are present. Cancer samples (n = 281), clinically tested by LiquidHALLMARK during Sep 2020-Sep 2021, and healthy samples (n = 28) were included for analysis. For each sample, fragment lengths were inferred from sequencing alignment files, and binned into “short” (0-150 bp) and “long” (151-500 bp) groups. A relative “size ratio” of the total number of short vs. long fragments per sample was calculated, and examined with clinical features, plasma cfDNA concentration (cfDNA/ml) and highest mutation allele frequency (AF%), in a model to predict cancer. Results: Calculated size ratios (relative abundance of short fragments) were higher in cancer than normal samples (median 47.6 vs. 31.2, p < 0.001). Cholangiocarcinoma and colorectal cancer samples had the highest size ratios (medians: 62.9 and 60.9, respectively) in agreement with a genome-wide NGS study that profiled cfDNA sizes. Size ratios were higher in metastatic (n = 143) compared to early stage (n = 30) lung cancers (p = 0.0039), indicating a stage-dependent accumulation of shorter cfDNA fragments. Size ratio was correlated with cfDNA/ml ( r= 0.63, p < 0.01) and AF% ( r= 0.42, p < 0.01). In 20-fold cross-validation of a logistic regression model trained to predict cancer, average area under curve (AUC) was 0.82 using size ratio, 0.86 using cfDNA/ml, and increased to 0.95 with the two features combined. Conclusions: Our analysis shows that it is feasible to derive meaningful cfDNA fragment size information from amplicon-based NGS data. Importantly, relative fragment size distributions observed in cancer and healthy plasma samples by this method are concordant with alternate target capture methods. Fragment size ratios derived from relatively small, targeted amplicon panels are a novel feature that, combined with other molecular and clinical features, can enhance non-invasive methods of cancer detection.

Fluorochrome Selection for Imaging Intraoperative Ovarian Cancer Probes.

The identification and removal of all gross and microscopic tumor to render the patient disease free represents a huge challenge in ovarian cancer treatment. The presence of residual disease is an independent negative prognostic factor. Herein, we describe the synthesis and the “in vitro” evaluation of compounds as cyclooxygenase (COX)-1 inhibitors, the COX-1 isoform being an ovarian cancer biomarker, each bearing fluorochromes with different fluorescence features. Two of these compounds N-[4-(9-dimethylimino-9H-benzo[a]phenoxazin-5-ylamino) butyl]-2-(3,4-bis(4-methoxyphenyl)isoxazol-5-yl)acetamide chloride (RR11) and 3-(6-(4-(2-(3,4-bis(4-methoxyphenyl)isoxazole-5-yl)acetamido)butyl)amino-6-oxohexyl)-2-[7-(1,3-dihydro-1,1-dimethyl-3-ethyl 2H-benz[e]indolin-2-yl-idene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-(6-carboxilato-hexyl)-1H-benz[e]indolium chloride, 23 (MSA14) were found to be potent and selective inhibitors of cyclooxygenase (COX)-1 “in vitro”, and thus were further investigated “in vivo”. The IC50 values were 0.032 and 0.087 µM for RR11 and 23 (MSA 14), respectively, whereas the COX-2 IC50 for RR11 is 2.4 µM while 23 (MSA14) did not inhibit COX-2 even at a 50 µM concentration. Together, this represented selectivity index = 75 and 874, respectively. Structure-based virtual screening (SBVS) performed with the Fingerprints for Ligands and Proteins (FLAP) software allowed both to differentiate highly active compounds from less active and inactive structures and to define their interactions inside the substrate-binding cavity of hCOX1. Fluorescent probes RR11 and 23 (MSA14), were used for preliminary near-infrared (NIR) fluorescent imaging (FLI) in human ovarian cancer (OVCAR-3 and SKOV-3) xenograft models. Surprisingly, a tumor-specific signal was observed for both tested fluorescent probes, even though this signal is not linked to the presence of COX-1.

NVD-BM-mediated genetic biosensor triggers accumulation of 7-dehydrocholesterol and inhibits melanoma via Akt1/NF-ĸB signaling.

Aberrant activation of the cholesterol biosynthesis supports tumor cell growth. In recent years, significant progress has been made by targeting rate-limiting enzymes in cholesterol biosynthesis pathways to prevent carcinogenesis. However, precise mechanisms behind cholesterol degradation in cancer cells have not been comprehensively investigated. Here, we report that codon optimization of the orthologous cholesterol 7-desaturase, NVD-BM from Bombyx mori, significantly slowed melanoma cell proliferation and migration, and inhibited cancer cell engraftment in nude mice, by converting cholesterol to toxic 7-dehydrocholesterol. Based on these observations, we established a synthetic genetic circuit to induce melanoma cell regression by sensing tumor specific signals in melanoma cells. The dual-input signals, RELA proto-oncogene (RELA) and signal transducer and activator of transcription 1 (STAT1), activated NVD-BM expression and repressed melanoma cell proliferation and migration. Mechanically, we observed that NVD-BM decreased Akt1-ser473 phosphorylation and inhibited cytoplasmic RELA translocation. Taken together, NVD-BM was identified as a tumor suppressor in malignant melanoma, and we established a dual-input biosensor to promote cancer cell regression, via Akt1/NF-κB signaling. Our results demonstrate the potential therapeutic effects of cholesterol 7-desaturase in melanoma metabolism, and provides insights for genetic circuits targeting 7-dehydrocholesterol accumulation in tumors.

Tumor-specific near-infrared nanobody probe rapidly labels tumors in an orthotopic mouse model of pancreatic cancer

BackgroundNanobodies, derived from camelid antibodies made of only heavy chains, are the smallest, biologic, antigen-binding fragments (~15kDa) with faster pharmacokinetics and better tumor penetration efficiency than standard antibodies. The present study evaluates the efficacy of a fluorescent, anti-carcinoembryonic antigen (CEA) nanobody for rapid tumor labeling in an orthotopic mouse model of pancreatic cancer. MethodsAnti-CEA or control nanobodies were conjugated with the near-infrared fluorophore IRDye 800CW. Fragments of BxPC-3 (high-CEA expressing) or MiaPACA-2 (low-CEA expressing) human pancreatic cancer cell lines were orthotopically implanted into the pancreatic tail of nude mice. After tumors reached 7 to 10 mm in size, 2 nmol anti-CEA or control nanobody-IRDye800CW were injected intravenously. Mice were imaged at various time points hours post-injection. ResultsAnti-CEA nanobodies clearly labeled BxPC3 orthotopic pancreatic tumors 3 hours after injection. The signal was present as early as 15 minutes after injection and was robust at 1 to 3 hours after injection with a tumor-to-background ratio of 2.66. In contrast, there was very low accumulation in the low CEA-expressing, MiaPACA2 pancreatic orthotopic tumors. The fluorophore-conjugated nanobody was specific for CEA-expressing tumors, while the control nanobody did not show any tumor-specific signal. Both nanobodies had strong kidney uptake as expected for small-molecule probes. The fluorescence signal was detectable using 2 clinical, Food and Drug Administration-approved, 800 nm imaging devices as well as small animal imaging systems. ConclusionThis anti-CEA, nanobody-based, fluorescent probe labeled pancreatic orthotopic tumors within 15 minutes of intravenous injection. Fluorescent anti-CEA nanobodies have labeling kinetics that approach the speed of nonspecific dyes such as indocyanine green but with the specificity of antibodies. The use of fluorescently-labeled, intact antibodies leads to a labeling delay of 48 to 96 hours between probe administration and the necessarily delayed time of operation, which can be avoided with nanobodies. The kinetics of a nanobody-based probe makes it a practical agent for same-day, patient administration and fluorescence-guided surgery.

A GSH-Gated DNA Nanodevice for Tumor-Specific Signal Amplification of microRNA and MR Imaging-Guided Theranostics.

Developing tumor-responsive diagnosis and therapy strategies for tumor theranostics is still a challenge owing to their high accuracy and specificity. Herein, an AND logic gated-DNA nanodevice, based on the fluorescence nucleic acid probe and polymer-modified MnO2 nanosheets, for glutathione (GSH)-gated miRNA-21 signal amplification and GSH-activated magnetic resonance (MR) imaging-guided chemodynamic therapy (CDT) is reported. In the presence of overexpressed miRNA and GSH (tumor cells), the nanodevice can be in situ activated and release significantly amplified fluorescence signals and MR signals. Conversely, the fluorescence signal is quenched and MR signal remains at the background level with low miRNA and GSH (normal cells), efficiently reducing the false-positive signals by more than 50%. Under the guide of miRNA profiling and MR imaging, the tumor-responsive hydroxyl radical (·OH) can effectively kill tumor cells. Furthermore, the nanodevice shows catalase-like activity and glucose oxidase-like activity with the performance of O2 production and glucose consumption. This is the first time to fabricate a tumor-responsive theranostic DNA nanodevice with tumor-specific signal amplification of microRNA and GSH-activated MR imaging for CDT, potential hypoxia relief and starvation therapy, which provides a new insight for designing smart theranostic strategies.

Near-infrared fluorescent guided surgery in patients with pancreatic cancer

Background: The irradical resection rate of pancreatic cancer (PC) worldwide is still ∼60% due to difficulties in discriminating tumor from normal tissue. Near-infrared fluorescence (NIRF) is a promising imaging technique, potentially improving intraoperative demarcation of PC and radical resection rates. In this study, a novel, fluorescent-labelled antibody targeting carcinoembryonic antigen (CEA), named SGM-101, was investigated. The tolerability and feasibility of intraoperative fluorescence tumor imaging using SGM-101 was assessed in patients undergoing a surgical exploration for pancreatic cancer. Methods: Patients (n = 12) were injected intravenously with 5, 7.5 or 10 mg SGM-101, 48, 72 or 96 hours prior to surgery. Tolerability assessment was performed at regular intervals after dosing. During surgery the Quest NIR imaging system was used to detect the fluorescent signals. The fluorescent signals of the resected specimens were correlated to the histopathological assessment. Results: SGM-101 specifically accumulated in primary tumors, peritoneal and liver metastases, with upregulated expression of CEA, allowing real-time intraoperative fluorescence imaging. Sufficient fluorescent signals were observed in primary tumors and in metastatic lesions. Of the resected lesions, one lesion was histologically assessed as benign but was fluorescent, two lesions were histologically assessed as malign but no fluorescent signal was detected. Conclusion: Our study demonstrates that SGM-101, a safe fluorescent-labelled anti-CEA antibody, is able to penetrate PC and allows intraoperative NIRF imaging. Both primary tumors and the metastases were intraoperatively detected. The current technique should be further improved to ib=increase tumor specific fluorescent signals and sensitivity.

PO-517 Peptide-based diagnostic in vivo targeting of membrane Hsp70 positive tumours

IntroductionHeat shock protein 70 (Hsp70, HSP1A1), the major stress-inducible stress response protein, is frequently overexpressed in tumour cells of many entities. Hsp70 plays a promotive role in the development, viability and metastatic spread of tumour cells, in vivo. In contrast to normal cells, next to its cytoplasmic localization Hsp70 is additionally found on the plasma membrane of many different tumour types. For tumour cell-selective in vivo targeting, we developed an membrane Hsp70 specific, peptide-based tumour targeting probe, termed ‘Tumour cell penetrating peptide’ (TPP).Material and methodsThe internalisation kinetics of the Hsp70 peptide tracer into tumour cells was determined by flow cytometry. The in vivotumour targeting capacity of fluorescence- or 89Zr radio-labelled TPP based probes were assessed by near infrared fluorescence imaging and PET/CT measurements, following intravenous administration of the compounds. For in vivo imaging, preclinical tumour models of syngeneic, spontaneous, chemically or genetically induced as well as xenografted tumours were used.Results and discussionsBy mimicking the oligomerization domain of Hsp70, binding specificity and affinity (Kd=19 nM) of TPP was achieved. Following the rapid intracellular turnover rate of membrane bound Hsp70, TPP is continuously internalised into tumour cells, leading to a rapid intracellular accumulation. In accordance, following intravenous administration in tumour bearing mice, the tracer is effectively protected from body clearance, which results in a high tumor-to-normal tissue signal ratio and precise tumour delineation within 24 hour after injection. In vivo, TPP accumulation reliably reflects the mHsp70 expression profiles of differently Hsp70 expressing tumour types. The peptide thereby differentiates between tumour cells and cells of the tumour microenvironment, such as tumor-associated macrophages and fbroblasts, which were found to be membrane-Hsp70 negative. Compared to a small molecule αvβ3-integrin antagonist probe, TPP based probes offer preferably high tumor-to-background contrast and tumor-specific signal intensity in the tested tumour models.ConclusionTPP provides a useful tool for multimodal imaging of tumours and metastases that might help to improve our understanding of tumour biology and allow the establishment of improved diagnostic procedures and more accurate therapeutic monitoring. TPP might also be a promising platform for tumor-specific drug delivery and other Hsp70- based targeted therapies.

Abstract IA18: Integrin-α10 drives metastasis and sarcomagenesis in mxyofibrosarcoma and undifferentiated pleomorphic sarcoma

Abstract Myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) are the most common and aggressive types of genetically complex sarcoma. The development of new targeted therapies is vital for improving patients' outcomes; however, the complexity of alterations in these sarcomas has made it difficult to find the true drivers of oncogenesis. To investigate the molecular basis of MFS and UPS oncogenesis and progression, we undertook gene expression and copy number profiling of a well-characterized set of 94 primary untreated high-grade MFS and UPS. Unsupervised clustering identified a subgroup associated with poor clinical outcome. This subgroup had strikingly higher expression of the integrin-α10 gene, ITGA10, and its expression was also independently associated with outcome. We therefore investigated the role of integrin-α10 in MFS and UPS cells, defined the pathways by which it exerts its effects, and tested pharmacologic inhibition of these pathways for antitumor effects both in vitro and in vivo. Silencing of integrin-α10 induces growth suppression through apoptosis in MFS and UPS cells but not in normal mesenchymal cells. Integrin-α10 transmitted its tumor-specific signal via TRIO and RICTOR, two oncoproteins that are frequently co-overexpressed through gene amplification on chromosome 5p. TRIO and RICTOR activated RAC/PAK and AKT/mTOR to promote sarcoma cell survival. Inhibition of these proteins with EHop-016 (RAC inhibitor) and INK128 (mTOR inhibitor) had antitumor effects in tumor-derived cell lines and mouse xenografts, and combining the drugs enhanced the effects. Our results demonstrate the importance of integrin-α10/TRIO/RICTOR signaling for driving myxofibrosarcoma progression and provide the basis for promising targeted treatment strategies for patients with high-risk disease. Citation Format: Samuel Singer. Integrin-α10 drives metastasis and sarcomagenesis in mxyofibrosarcoma and undifferentiated pleomorphic sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA18.

In vivo cation exchange in quantum dots for tumor-specific imaging

In vivo tumor imaging with nanoprobes suffers from poor tumor specificity. Here, we introduce a nanosystem, which allows selective background quenching to gain exceptionally tumor-specific signals. The system uses near-infrared quantum dots and a membrane-impermeable etchant, which serves as a cation donor. The etchant rapidly quenches the quantum dots through cation exchange (ionic etching), and facilitates renal clearance of metal ions released from the quantum dots. The quantum dots are intravenously delivered into orthotopic breast and pancreas tumors in mice by using the tumor-penetrating iRGD peptide. Subsequent etching quenches excess quantum dots, leaving a highly tumor-specific signal provided by the intact quantum dots remaining in the extravascular tumor cells and fibroblasts. No toxicity is noted. The system also facilitates the detection of peritoneal tumors with high specificity upon intraperitoneal tumor targeting and selective etching of excess untargeted quantum dots. In vivo cation exchange may be a promising strategy to enhance specificity of tumor imaging.

PH-Triggered and Enhanced Simultaneous Photodynamic and Photothermal Therapy Guided by Photoacoustic and Photothermal Imaging

Developing smart photosensitizers sensitively responding to tumor-specific signals for reduced side effects and enhanced anticancer efficacy is a major challenge for tumor phototherapy. Herein, a pH-sensitive photosensitizer has been synthesized through introducing a pH-sensitive receptor (dimethylaminophenyl unit) onto the aza-BODIPY core (abbreviated as NAB). Through enveloping hydrophobic NAB with amphiphilic DSPE-mPEG2000, NAB nanoparticles (NPs, diameter ∼ 30 nm) with strong near-infrared absorption (∼792 nm) are obtained. NAB NPs can be activated in weak acidic environment to give high rate of reactive oxygen species (ROS) generation and enhanced photothermal effect. NAB NPs can selectively accumulate in the lysosomes of tumor cells and subsequently activate under the acidic microenvironment of lysosome (pH 5.0) to produce ROS for photodynamic therapy, due to switch-off of the photoinduced electron transfer (PET) pathway. In vivo, pH-enhanced photoacoustic imaging (PAI) and photothermal imaging (PTI...

Tumor antigen–specific T cells for immune monitoring of dendritic cell–treated glioblastoma patients

Background aimsCD8+ T cells are part of the adaptive immune system and, as such, are responsible for the elimination of tumor cells. Dendritic cells (DC) are professional antigen-presenting cells (APC) that activate CD8+ T cells. Effector CD8+ T cells in turn mediate the active immunotherapeutic response of DC vaccination against the aggressive glioblastoma (GBM). The lack of tumor response assays complicates the assessment of treatment success in GBM patients. MethodsA novel assay to identify specific cytotoxicity of activated T cells by APC was evaluated. Tumor antigen-pulsed DCs from HLA-A*02-positive GBM patients were cultivated to stimulate autologous cytotoxic T lymphocytes (CTL) over a 12-day culture period. To directly correlate antigen specificity and cytotoxic capacity, intracellular interferon (IFN)-γ fluorescence flow cytometry-based measurements were combined with anti-GBM tumor peptide dextramer staining. IFN-γ response was quantified by real-time polymerase chain reaction (PCR), and selected GBM genes were compared with healthy human brain cDNA by single specific primer PCR characterization. ResultsUsing CTL of GBM patients stimulated with GBM lysate-pulsed DCs increased IFN-γ messenger RNA levels, and intracellular IFN-γ protein expression was positively correlated with specificity against GBM antigens. Moreover, the GBM peptide-specific CD8+ T-cell response correlated with specific GBM gene expression. Following DC vaccination, GBM patients showed 10-fold higher tumor-specific signals compared with unvaccinated GBM patients. DiscussionThese data indicate that GBM tumor peptide-dextramer staining of CTL in combination with intracellular IFN-γ staining may be a useful tool to acquire information on whether a specific tumor antigen has the potential to induce an immune response in vivo.

320. Adenovirus Expansion of Radioiodine Therapy to Non-Thyroid Cancer Treatment and Diagnostics

Radioiodine therapy has successfully treated and diagnosed cancers over the last 50 years. The clinical scope of radioiodine is limited in thyroid cancers because of restricted iodine uptake. Our lab proposes to expand the clinical use of radioiodine into non-thyroid cancer theranostics by targeting cancers (e.g. pancreatic and prostate) with oncolytic adenovirus to over express the human sodium iodine symporter (NIS). The adenovirus expression of the symporter allows iodine to concentrate into the targeted cancer cells providing diagnostic imaging with I123 and therapy with I131.We designed infectivity-enhanced oncolytic Adenoviruses (NIS-OAd), Ad5/3_TSP_E3_ADP_NIS, to express NIS and adenovirus death protein (ADP) from the E3 region. The Ad5/3 fiber modification enables the infection of CAR negative cells. Vector replication is controlled through a tumor specific promoter (TSP, e.g. COX2 in pancreatic and Pb in prostate cancer). We have shown the specificity of our viruses with TSPs in vitro with multiple pancreatic and prostate cancer cell lines. The expression of ADP resulted in an increase in oncolytic activity and transgene expression. The functionality of NIS-OAds was tested with in vitro radioiodine uptake assay and showed up to a 10-fold increase in radioiodine uptake in prostate and pancreatic cancer cell lines. This endorses that oncolytic viruses increase functional NIS expression in tumors.Our animal model experiments included nude mice xenographs to evaluate therapeutic and imaging affects with i.v. injections of NIS-OAds in A549 lung and LnCap prostate cancer cells. In A549 models, the combination of NIS-OAds and I131 had an increase in therapeutic effect over virus alone signifying functional NIS expression in solid tumors. LnCap xenographs had SPECT/CT images taken after NIS-OAds and non-replicating virus administration with I123. NIS-OAds had the highest tumor specific signal confirming that NIS-OAds can be diagnostic in tumors.Viral functionality in human tissue was evaluated by PCR in ex vivo tissue slices showing that virus replication was specific to cancerous tissues and did not occur in normal tissue. This study supports the high-level replication control of NIS-OAds in non-thyroid cancer tissues. A related non-replicating NIS expressing Ad is also being evaluated in prostate cancer by Dr. Morris, our collaborator, in a stage I clinical trial. Together the virus specificity, imaging, and therapeutic data support the potential of NIS-OAds as cancer theranostics.In sum, this data provides key functionality and safety support of NIS expressing Ad use in humans. Current projects include therapeutic and imaging animal studies with radioiodine in pancreatic cancer immuno-competent Syrian hamsters, and human xenographs in nude mice. Further studies will evaluate the distribution of NIS expression in vivo and cytotoxicity of virus with radiation. Ultimately the goal of our research is to form a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.

Time-Frequency Analysis of Peptide Microarray Data: Application to Brain Cancer Immunosignatures.

One of the gravest dangers facing cancer patients is an extended symptom-free lull between tumor initiation and the first diagnosis. Detection of tumors is critical for effective intervention. Using the body’s immune system to detect and amplify tumor-specific signals may enable detection of cancer using an inexpensive immunoassay. Immunosignatures are one such assay: they provide a map of antibody interactions with random-sequence peptides. They enable detection of disease-specific patterns using classic train/test methods. However, to date, very little effort has gone into extracting information from the sequence of peptides that interact with disease-specific antibodies. Because it is difficult to represent all possible antigen peptides in a microarray format, we chose to synthesize only 330,000 peptides on a single immunosignature microarray. The 330,000 random-sequence peptides on the microarray represent 83% of all tetramers and 27% of all pentamers, creating an unbiased but substantial gap in the coverage of total sequence space. We therefore chose to examine many relatively short motifs from these random-sequence peptides. Time-variant analysis of recurrent subsequences provided a means to dissect amino acid sequences from the peptides while simultaneously retaining the antibody–peptide binding intensities. We first used a simple experiment in which monoclonal antibodies with known linear epitopes were exposed to these random-sequence peptides, and their binding intensities were used to create our algorithm. We then demonstrated the performance of the proposed algorithm by examining immunosignatures from patients with Glioblastoma multiformae (GBM), an aggressive form of brain cancer. Eight different frameshift targets were identified from the random-sequence peptides using this technique. If immune-reactive antigens can be identified using a relatively simple immune assay, it might enable a diagnostic test with sufficient sensitivity to detect tumors in a clinically useful way.

Selective in vivo imaging of syngeneic, spontaneous, and xenograft tumors using a novel tumor cell-specific hsp70 peptide-based probe.

Although in vivo targeting of tumors using fluorescently labeled probes has greatly gained in importance over the last few years, most of the clinically applied reagents lack tumor cell specificity. Our novel tumor cell-penetrating peptide-based probe (TPP) recognizes an epitope of Hsp70 that is exclusively present on the cell surface of a broad variety of human and mouse tumors and metastases, but not on normal tissues. Because of the rapid turnover rate of membrane Hsp70, fluorescently labeled TPP is continuously internalized into syngeneic, spontaneous, chemically/genetically induced and xenograft tumors following intravenous administration, thereby enabling site-specific labeling of primary tumors and metastases. In contrast with the commercially available nonpeptide small molecule αvβ3-integrin antagonist IntegriSense, TPP exhibits a significantly higher tumor-to-background contrast and stronger tumor-specific signal intensity in all tested tumor models. Moreover, in contrast with IntegriSense, TPP reliably differentiates between tumor cells and cells of the tumor microenvironment, such as tumor-associated macrophages and fibroblasts, which were found to be membrane-Hsp70 negative. Therefore, TPP provides a useful tool for multimodal imaging of tumors and metastases that might help to improve our understanding of tumorigenesis and allow the establishment of improved diagnostic procedures and more accurate therapeutic monitoring. TPP might also be a promising platform for tumor-specific drug delivery and other Hsp70-based targeted therapies.

Abstract 687: A comparative study of p95-HER2 carboxy terminal fragment (CTF) detected by immunohistochemistry and VeraTag assays in human breast tumors

Abstract C-terminally truncated forms of HER2 (HER2-CTF) have been reported to be highly oncogenic and correlate with node-positive breast cancer, poor prognosis, and attenuated response to trastuzumab in patients with HER2+ metastatic breast cancer (MBC). A quantitative, sensitive and specific immunoassay using the VeraTag™ technology has been developed to detect the active Met611-HER2 CTF (p95) in FFPE tumor samples and has been used to identify HER2+ MBC patients who have a reduced response to trastuzumab in two independent cohorts. Recently, an IHC assay using an antibody directed to a similar p95 epitope was reported to detect a high percentage of HER2+ samples (79%) as p95 positive (#530, ASCO 2011). To verify the cellular specificity and accuracy of the VeraTag p95 assay, and evaluate the prevalence of p95 in breast tumors, an IHC assay was developed which utilizes the same p95 antibody as the VeraTag assay. The IHC and VeraTag assay formats were compared in 120 human breast tumor excisional and core biopsy samples submitted to central clinical reference lab testing, and having different levels of HER2 measured by HercepTest IHC: 0 (n=20); 1+ (n=20), 2+ (n=20), and 3+ (n=60). Replicate slides were run in both VeraTag and IHC assays to demonstrate reproducibility. Nearly all p95 IHC signal was associated with tumor cells only. Consistent with earlier VeraTag p95 data, a general correlation was observed between p95 and HER2 levels, with tumors expressing normal levels of HER2 rarely expressing high p95 levels. However, within the subset of HER2+ samples (IHC 3+ or FISH > 2.2; n=63), a ∼30-fold range of p95 VeraTag signal was observed, with 44% having p95 expression above the level of a previously determined clinical cutoff for trastuzumab response (Clin Cancer Res 16: 4226 2010). A similar relationship was observed for samples assayed by p95 IHC. All samples measured as p95 IHC 3+ (>10% cells with 3+ membranous staining) were HER2+ by central lab testing and represented nearly a third of the HER2+ samples. The VeraTag p95 assay generally agreed with p95 IHC scored by test category 0-3+ (r=0.54; p<0.0001) or histoscore (r=0.54; p<0.0001) with 79% of the p95 IHC 3+ samples falling above the p95 VeraTag assay clinical cutoff. Fewer p95 VeraTag positive samples were represented in p95 IHC 2+ and 1+ categories. These results indicate that the tumor-specific p95 signal detected by the p95 VeraTag assay is in general agreement with an IHC assay using the same antibody; however a number of cases with p95 VeraTag scores above the clinical cutoff do not fall into the p95 IHC 3+ category. Additionally, the prevalence of p95 positivity by VeraTag assay and by IHC 3+ scores is consistent with earlier peer-reviewed studies and lower than recently reported using a different IHC assay. The clinical significance of using the p95 IHC assay to complement the VeraTag p95 score is being evaluated. 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 687. doi:1538-7445.AM2012-687

Molecular Alternations in Uveal Melanoma

The eye as an immune privileged organ is mostly spared from (auto)immune attacks. Intraocular inflammation like autoimmune uveitis is a rare event. Nevertheless, tumor-related destructive autoimmune responses can affect the eye, as observed in the case of cancer- associated retinopathy (CAR), an autoantibody-mediated destruction of retinal cells induced by the ectopic expression of ocular antigens by peripheral tumors. The new tumor therapies targeting immune checkpoints to enhance anti-tumor responses can also induce autoimmune responses and result in autoimmune diseases even in immune privileged organs like the eyes. Even MEK/BRAF-inhibitor therapies using small molecules to block tumor-specific signal transduction molecules have turned out to not just inhibit tumor growth and survival and render tumors more susceptible for immune recognition, but to have additional toxic effects on non-dividing retinal cells, destroying and making them potential targets of autoimmunity.

Gadolinium Chloride Augments Tumor-Specific Imaging of Targeted Quantum Dots In Vivo

Nonspecific sequestration of nanoparticles by the reticulo-endothelial system (RES) results in the degradation of image quality of nanoparticle-based imaging. We demonstrate that gadolinium chloride (GdCl3) pretreatment inactivates RES macrophages, thereby increasing circulatory time and amplifying the tumor-specific signal of conjugated nanoparticles in vivo. The experimental results were validated using compartmental modeling, and the rate parameters for the observed kinetics pattern were estimated. This pretreatment strategy could have broad applicability across biomedical applications utilizing theranostic nanoparticles that are sequestered by the RES.