Martin Auerbach

Adoptive Transfer of MART-1 T-Cell Receptor Transgenic Lymphocytes and Dendritic Cell Vaccination in Patients with Metastatic Melanoma

Purpose: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy. Experimental Design: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation. Results: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1–specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1–specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells. Conclusion: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. Clin Cancer Res; 20(9); 2457–65. ©2014 AACR.

18F-FDOPA PET and PET/CT Accurately Localize Pheochromocytomas

Successful treatment of pheochromocytoma requires accurate diagnosis and localization of tumors. Herein, we investigated the accuracy of PET using 3,4-dihydroxy-6-18F-fluoro-phenylalanine (18F-FDOPA), an amino acid transporter substrate, as an independent marker for detection of benign and malignant pheochromocytomas. Methods: The study comprised 25 consecutive patients (9 men, 16 women) whose median age was 51 y (range, 25–68 y), with known or suspected pheochromocytoma. Eleven patients underwent standardized 18F-FDOPA PET and 14 patients underwent 18F-FDOPA PET/CT studies, with a median of 511 MBq of 18F-FDOPA (range, 206–625 MBq). Two readers, unaware of the reports of other imaging studies and clinical data, analyzed all scans visually and quantitatively (maximum standardized uptake value [SUVmax] and maximum transverse diameter). Histology and long-term clinical follow-up served as the gold standard. Correlation between SUVmax of tumors and biochemical markers was evaluated. SUVmax of the benign and malignant tumors was compared. Results: Seventeen patients underwent surgery. Histology confirmed pheochromocytoma or paraganglioma in 11 cases (8 adrenal, including 2 malignant tumors, and 3 extraadrenal, including 1 malignant tumor). The diagnosis of pheochromocytoma was established by follow-up in 2 additional patients (1 adrenal and 1 unknown location) and ruled out in 6 patients. Visual analysis detected and localized pheochromocytoma in 11 of 13 patients without false-positive results (sensitivity, 84.6%; specificity, 100%; accuracy, 92%). These lesions had an SUVmax of 2.3–34.9 (median, 8.3). Evaluation of the false-negative cases revealed a 13 × 5 mm lesion with an SUVmax of 1.96 in 1 case; no lesion was localized in the second case using multiple additional modalities. Spearman nonparametric analysis did not show statistically significant correlation between SUVmax of the tumors and biochemical markers. The Mann–Whitney nonparametric test did not demonstrate a statistically significant difference between the SUVmax of 18F-FDOPA in malignant and benign tumors. Conclusion: 18F-FDOPA PET and PET/CT are highly sensitive and specific tools that can provide additional independent information for diagnosis and localization of benign and malignant pheochromocytomas.

Structure-guided Engineering of Human Thymidine Kinase 2 as a Positron Emission Tomography Reporter Gene for Enhanced Phosphorylation of Non-natural Thymidine Analog Reporter Probe

Background: Humanized PET reporter gene (PRG) systems are needed to replace immunogenic, viral-derived systems. Results: Employing a structure-guided approach, we developed a highly sensitive humanized PRG characterized by reduced activity for its natural substrates. Conclusion: Sensitivity of PRGs can be improved by reducing their endogenous activities. Significance: Our method can be employed to rapidly develop highly sensitive humanized PRGs. Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2′-deoxy-2′-18F-5-methyl-1-β-l-arabinofuranosyluracil (l-18F-FMAU) as the PRP. We identified l-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates l-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/l-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Association of positive dual-modality positron emission tomography/computed tomography imaging of primary choroidal melanoma with chromosome 3 loss and tumor size.

Purpose: To evaluate the positive dual-modality positron emission tomography/computed tomography (PET/CT) of choroidal melanoma with chromosome 3 loss and tumor size. Methods: Thirty-seven consecutive patients with choroidal melanoma with known chromosome 3 status who underwent whole-body PET/CT imaging were retrospectively reviewed. Cytology and chromosome 3 loss were identified by fine-needle aspiration biopsy. Fluorescent in situ hybridization and whole genome microarray by single-nucleotide polymorphism were used to evaluate the chromosome 3 status. Metabolic activity of primary choroidal melanoma by PET/CT imaging was evaluated. Results: Thirteen of 37 (35%) primary choroidal melanomas had loss of chromosome 3; 7 of the 13 (54%) melanomas were positive for metabolic activity identified by PET/CT imaging. All 24 of 37 melanomas without chromosome 3 loss were inactive for metabolic activity. There was a statistically significant association between positive metabolic activity and chromosome 3 loss (P = 0.00017 Fisher exact test); positive PET/CT imaging was 54% sensitive and 100% specific for loss of chromosome 3. Seven of 37 (19%) choroidal melanomas with positive metabolic activity by PET/CT were statistically significantly larger in size than the 30 metabolically inactive melanomas (P < 0.001, Kruskal-Wallis test). Conclusion: Positive metabolic activity of choroidal melanoma identified by PET/CT imaging was statistically significantly associated with chromosome 3 loss and larger tumor size.

Preliminary results of automated removal of degenerative joint disease in bone scan lesion segmentation

Whole-body bone scintigraphy (or bone scan) is a highly sensitive method for visualizing bone metastases and is the accepted standard imaging modality for detection of metastases and assessment of treatment outcomes. The development of a quantitative biomarker using computer-aided detection on bone scans for treatment response assessment may have a significant impact on the evaluation of novel oncologic drugs directed at bone metastases. One of the challenges to lesion segmentation on bone scans is the non-specificity of the radiotracer, manifesting as high activity related to non-malignant processes like degenerative joint disease, sinuses, kidneys, thyroid and bladder. In this paper, we developed an automated bone scan lesion segmentation method that implements intensity normalization, a two-threshold model, and automated detection and removal of areas consistent with non-malignant processes from the segmentation. The two-threshold model serves to account for outlier bone scans with elevated and diffuse intensity distributions. Parameters to remove degenerative joint disease were trained using a multi-start Nelder-Mead simplex optimization scheme. The segmentation reference standard was constructed manually by a panel of physicians. We compared the performance of the proposed method against a previously published method. The results of a two-fold cross validation show that the overlap ratio improved in 67.0% of scans, with an average improvement of 5.1% points.

Automated segmentation of tumors on bone scans using anatomy-specific thresholding

Quantification of overall tumor area on bone scans may be a potential biomarker for treatment response assessment and has, to date, not been investigated. Segmentation of bone metastases on bone scans is a fundamental step for this response marker. In this paper, we propose a fully automated computerized method for the segmentation of bone metastases on bone scans, taking into account characteristics of different anatomic regions. A scan is first segmented into anatomic regions via an atlas-based segmentation procedure, which involves non-rigidly registering a labeled atlas scan to the patient scan. Next, an intensity normalization method is applied to account for varying levels of radiotracer dosing levels and scan timing. Lastly, lesions are segmented via anatomic regionspecific intensity thresholding. Thresholds are chosen by receiver operating characteristic (ROC) curve analysis against manual contouring by board certified nuclear medicine physicians. A leave-one-out cross validation of our method on a set of 39 bone scans with metastases marked by 2 board-certified nuclear medicine physicians yielded a median sensitivity of 95.5%, and specificity of 93.9%. Our method was compared with a global intensity thresholding method. The results show a comparable sensitivity and significantly improved overall specificity, with a p-value of 0.0069.