Khun Visith Keu

Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma

PET gene reporter imaging can be used to monitor the trafficking of therapeutic cytotoxic T cells in glioma patients. T cells reporting for duty Cytotoxic T cells engineered to kill tumor cells are becoming a mainstay of cancer immunotherapy. However, no matter how precisely they are engineered, once they are injected into a patient, they are no longer directly monitored or controlled by the researchers. As a result, if the treatment fails to work or causes toxicity, it is not clear whether the therapeutic cells are ineffective or whether they scattered through normal tissues and never reached the tumor. Keu et al. have designed a method to engineer these T cells with a reporter gene such that they can be tracked in people by positron emission tomography. The authors present a clinical trial demonstrating the feasibility and safety of this approach in glioma patients. High-grade gliomas are aggressive cancers that often become rapidly fatal. Immunotherapy using CD8+ cytotoxic T lymphocytes (CTLs), engineered to express both herpes simplex virus type 1 thymidine kinase (HSV1-TK) and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR), is a treatment strategy with considerable potential. To optimize this and related immunotherapies, it would be helpful to monitor CTL viability and trafficking to glioma cells. We show that noninvasive positron emission tomography (PET) imaging with 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG) can track HSV1-tk reporter gene expression present in CAR-engineered CTLs. [18F]FHBG imaging was safe and enabled the longitudinal imaging of T cells stably transfected with a PET reporter gene in patients. Further optimization of this imaging approach for monitoring in vivo cell trafficking should greatly benefit various cell-based therapies for cancer.

Circulating Tumor Microemboli Diagnostics for Patients with Non–Small-Cell Lung Cancer

Introduction: Circulating tumor microemboli (CTM) are potentially important cancer biomarkers, but using them for cancer detection in early-stage disease has been assay limited. We examined CTM test performance using a sensitive detection platform to identify stage I non–small-cell lung cancer (NSCLC) patients undergoing imaging evaluation. Methods: First, we prospectively enrolled patients during 18F-FDG PET-CT imaging evaluation for lung cancer that underwent routine phlebotomy where CTM and circulating tumor cells (CTCs) were identified in blood using nuclear (DAPI), cytokeratin (CK), and CD45 immune-fluorescent antibodies followed by morphologic identification. Second, CTM and CTC data were integrated with patient (age, gender, smoking, and cancer history) and imaging (tumor diameter, location in lung, and maximum standard uptake value [SUVmax]) data to develop and test multiple logistic regression models using a case-control design in a training and test cohort followed by cross-validation in the entire group. Results: We examined 104 patients with NSCLC, and the subgroup of 80 with stage I disease, and compared them to 25 patients with benign disease. Clinical and imaging data alone were moderately discriminating for all comers (Area under the Curve [AUC] = 0.77) and by stage I disease only (AUC = 0.77). However, the presence of CTM combined with clinical and imaging data was significantly discriminating for diagnostic accuracy in all NSCLC patients (AUC = 0.88, p value = 0.001) and for stage I patients alone (AUC = 0.87, p value = 0.002). Conclusion: CTM may add utility for lung cancer diagnosis during imaging evaluation using a sensitive detection platform.

An Observational Study of Circulating Tumor Cells and 18F-FDG PET Uptake in Patients with Treatment-Naive Non-Small Cell Lung Cancer

Introduction We investigated the relationship of circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) with tumor glucose metabolism as defined by 18F-fluorodeoxyglucose (FDG) uptake since both have been associated with patient prognosis. Materials & Methods We performed a retrospective screen of patients at four medical centers who underwent FDG PET-CT imaging and phlebotomy prior to a therapeutic intervention for NSCLC. We used an Epithelial Cell Adhesion Molecule (EpCAM) independent fluid biopsy based on cell morphology for CTC detection and enumeration (defined here as High Definition CTCs or “HD-CTCs”). We then correlated HD-CTCs with quantitative FDG uptake image data calibrated across centers in a cross-sectional analysis. Results We assessed seventy-one NSCLC patients whose median tumor size was 2.8 cm (interquartile range, IQR, 2.0–3.6) and median maximum standardized uptake value (SUVmax) was 7.2 (IQR 3.7–15.5). More than 2 HD-CTCs were detected in 63% of patients, whether across all stages (45 of 71) or in stage I disease (27 of 43). HD-CTCs were weakly correlated with partial volume corrected tumor SUVmax (r = 0.27, p-value = 0.03) and not correlated with tumor diameter (r = 0.07; p-value = 0.60). For a given partial volume corrected SUVmax or tumor diameter there was a wide range of detected HD-CTCs in circulation for both early and late stage disease. Conclusions CTCs are detected frequently in early-stage NSCLC using a non-EpCAM mediated approach with a wide range noted for a given level of FDG uptake or tumor size. Integrating potentially complementary biomarkers like these with traditional patient data may eventually enhance our understanding of clinical, in vivo tumor biology in the early stages of this deadly disease.

The Clinical Use of PET/CT in the Evaluation of Melanoma

Positron emission tomography combined with computed tomography (PET/CT) has emerged in the last decade as a dominant imaging modality used for staging, monitoring response and surveillance of various cancers, including melanoma. Using 2-deoxy-2-((18)F)fluoro-D-glucose ((18)F-FDG) as the radiopharmaceutical, PET/CT has demonstrated its efficacy and its utility in the management of patients with advanced melanoma. Nonetheless, challenges remain in the early stage evaluation of melanoma and in the development of novel radiotracers to better characterize lesions found on PET/CT. This chapter focuses on the advantages and limitations of this imaging modality in melanoma. We also detail and describe the approach to perform (18)F-FDG PET/CT, the methods to accurately quantify lesions, as well as the pearls/pitfalls of image interpretation. Finally, an overview of preclinical and investigational clinical radiopharmaceuticals is presented.

Pilot Preclinical and Clinical Evaluation of (4S)-4-(3-[18F]Fluoropropyl)-L-Glutamate (18F-FSPG) for PET/CT Imaging of Intracranial Malignancies

Purpose (S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid (18F-FSPG) is a novel radiopharmaceutical for Positron Emission Tomography (PET) imaging. It is a glutamate analogue that can be used to measure xC- transporter activity. This study was performed to assess the feasibility of 18F-FSPG for imaging orthotopic brain tumors in small animals and the translation of this approach in human subjects with intracranial malignancies. Experimental Design For the small animal study, GS9L glioblastoma cells were implanted into brains of Fischer rats and studied with 18F-FSPG, the 18F-labeled glucose derivative 18F-FDG and with the 18F-labeled amino acid derivative 18F-FET. For the human study, five subjects with either primary or metastatic brain cancer were recruited (mean age 50.4 years). After injection of 300 MBq of 18F-FSPG, 3 whole-body PET/Computed Tomography (CT) scans were obtained and safety parameters were measured. The three subjects with brain metastases also had an 18F-FDG PET/CT scan. Quantitative and qualitative comparison of the scans was performed to assess kinetics, biodistribution, and relative efficacy of the tracers. Results In the small animals, the orthotopic brain tumors were visualized well with 18F-FSPG. The high tumor uptake of 18F-FSPG in the GS9L model and the absence of background signal led to good tumor visualization with high contrast (tumor/brain ratio: 32.7). 18F-FDG and 18F-FET showed T/B ratios of 1.7 and 2.8, respectively. In the human pilot study, 18F-FSPG was well tolerated and there was similar distribution in all patients. All malignant lesions were positive with 18F-FSPG except for one low-grade primary brain tumor. In the 18F-FSPG-PET-positive tumors a similar T/B ratio was observed as in the animal model. Conclusions 18F-FSPG is a novel PET radiopharmaceutical that demonstrates good uptake in both small animal and human studies of intracranial malignancies. Future studies on larger numbers of subjects and a wider array of brain tumors are planned. Trial Registration ClinicalTrials.gov NCT01186601

The great mimicker: an extensive bone sarcoidosis.

Sarcoidosis, a multisystem disease characterized by the formation of noncaseating granulomas, occurs classically in the lungs, skin, hilar lymph nodes and eyes, although any organ can be infiltrated [1, 2]. The bones are affected in 1–13 % of patients, as osteolytic, osteoporotic or cystic lesions [3, 4]. Bone sarcoidosis is most frequently found in the peripheral skeleton such as the phalanges, metacarpals and metatarsals [3, 4]. A 59-year-old woman presented with severe lower-back pain for the past 3 months. An initial CT scan revealed multiple mixed bone lesions, resembling an impressivemetastatic pattern. F-FDG PET/CT was performed in search of a primary cancer. FDG uptake revealed a myriad of hypermetabolic bone lesions (SUVmax 11.3) located throughout the axial and proximal appendicular skeleton (a–c) and predominantly osteolytic in nature. A hypermetabolic lesion (SUVmax 11.1) was also discovered in the uterine fundus (b). As accessory findings, there were several pulmonary acinar opacities and faintly hypermetabolic calcified hilar, mediastinal and paratracheal lymph nodes. Bone biopsies revealed fibrosis (d) and noncaseating granulomatous infiltration (e) of the bone marrow, compatible with extensive bone sarcoidosis. Special stains and immunohistochemistry eliminated other diagnoses. The patient underwent total hysterectomy and bilateral salpingo-oophorectomy, confirming the simultaneous diagnosis of an endometrioid adenocarcinoma. The primary interest of this image is the extensive bone distribution of sarcoidosis, in the context of a newly discovered cancer, mimicking a metastatic pattern. Although bone

Classic biphasic pulmonary blastoma demonstrated by 18F-FDG PET/CT.

A 75-year-old nonsmoker woman was referred for the evaluation of a nonsecretory left adrenal lesion. An abdominal contrast-enhanced CT showed an incidental left lower lobe mass, which was confirmed on a chest contrast-enhanced CT. A 18F-FDG PET/CT showed a hypermetabolic tumor without nodal or distant metastasis. She underwent a lobectomy, and the final pathology reported a classic biphasic pulmonary blastoma, which is an uncommon histological form of malignant lung neoplasm. This case highlighted the glucose avidity of this rare and aggressive cancer.

Liver Trapping of (99m)Tc Macroaggregated Albumin During Ventilation/Perfusion Scintigraphy in a Patient With Superior Vena Cava Stenosis as Demonstrated by SPECT/CT.

A 50-year-old woman presented to our institution with a 1-day history of right posterior thoracic pain and dyspnea. She had a previous history of conservative resection of a high-grade basal-like infiltrating ductal carcinoma of the right breast 2 years before, subsequently treated by chemotherapy and radiotherapy. A ventilation and perfusion (VQ) scintigraphy performed for suspected pulmonary embolism showed an abnormal deposition of (99m)Tc macroaggregated albumin ((99m)Tc-MAA) in the left lobe of the liver. This unusual finding prompted additional imaging that demonstrated a superior vena cava stenosis.

Cold Spondylodiscitis on 18F-FDG PET/CT in a Patient With Myelofibrosis.

A 74-year-old man was evaluated by bone scan for possible rib fractures, which revealed a diffuse skeletal uptake secondary to myelofibrosis and focal midthoracic uptake. Chest CT and F-FDG PET/CT demonstrated pathological vertebral fractures of the midthoracic spine, which appeared as a cold defect on PET comparing to intense marrow uptake related to myelofibrosis and a lung nodule with a low uptake, later biopsy-proven as non-small cell lung carcinoma (NSCLC). MRI confirmed CT findings for the spine noting also an enhanced epidural collection. Biopsies did not reveal malignant cells. Final diagnosis was spondylodiscitis and a stage I NSCLC.

Abstract 3485: Developing a non-invasive, diagnostic test for stage I non-small cell lung cancer using circulating tumor cells.

Introduction: Reliable, non-invasive diagnostic blood tests to assess high-risk pulmonary nodules are needed to avoid unnecessary invasive procedures and the risk of missing lung cancer. Methods: We used a prospective, observational, case-control study design of consecutively enrolled patients undergoing imaging for a lung nodule or diagnosed malignancy to investigate whether a non-EpCAM based circulating tumor cell (CTC) platform could accurately diagnose stage I non-small cell lung cancer (NSCLC). Phlebotomy was performed according to standardized protocols at all three participating medical centers and the number of CTCs was analyzed in a single blinded fashion on a continuous scale per mL of blood. For determining test characteristics, cases consisted of patients with stage I NSCLC, and controls consisted of advanced NSCLC, malignant non-NSCLC lung nodules, and benign lung nodules. Differences by disease group were compared formally by ANOVA or Chi-square analysis, and test performance for identifying stage I NSCLC compared to benign disease was assessed by sensitivity, specificity and C statistics. Results: Seventy-three of 79 samples were adequate for analysis after processing. The median age was 69 years (interquartile range [IQR] 65-76), 73% were male, 70% were Caucasian, 79% had a smoking history, and 45% had a history of cancer. Diagnoses consisted of 56 NSCLC patients, of which two-thirds were adenocarcinoma and 45 were stage I, 8 malignant non-NSCLC cases, and 9 benign nodules. There were no differences in basic clinical characteristics across diagnosis type other than plasma white blood cell count. Average nodule diameter was 2.2 cm (IQR 1.6-2.8) and this did not vary significantly by diagnosis. Median CTC/ml was 3.3, 3.0, 0.60 and 0.70 for Stage I NSCLC, Stage II-IV NSCLC, other non-NSCLCs, and benign nodules respectively. At a threshold of 1.0 CTC/mL, 2.0 CTC/mL and 5.0 CTC/mL respectively 63%, 54% and 44% of stage I NSCLCs had a detectable CTC burden. For these three thresholds sensitivity (89%, 89%, 100%) and specificity (64%, 53%, 44%) was calculated for discriminating stage I NSCLC from benign disease. The accuracy (C-statistic) for identifying stage I disease only relative to benign disease only improved from 0.84 to 0.91 when adding CTC/mL data to age, gender, smoking and cancer history, nodule size and location, and SUVmax. Discussion: CTCs may be useful for identifying stage I NSCLC from benign lung nodules. Addition of these data to non-invasive clinical and imaging parameters we currently use to risk stratify patients could improve clinical diagnosis for this group. Citation Format: Madelyn S. Luttgen, Khun Visith Keu, Viswam S. Nair, George Horng, Minal Vasanawala, Anand Kolatkar, Anders Carlsson, Mohsen Sabouri, Billy W. Loo, Joseph B. Shrager, Andrei Iagaru, Ware Kuschner, Peter Kuhn, Sanjiv S. Gambhir. Developing a non-invasive, diagnostic test for stage I non-small cell lung cancer using circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2013-3485