Eur J Nucl Med Mol Imaging (2015) 42:537–561 DOI 10.1007/s00259-014-2984-3 REVIEW ARTICLE Radiopharmaceuticals as probes to characterize tumour tissue Israt S. Alam & Mubarik A. Arshad & Quang-Dé Nguyen & Eric O. Aboagye Received: 17 December 2014 / Accepted: 18 December 2014 / Published online: 3 February 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Tumour cells exhibit several properties that allow them to grow and divide. A number of these properties are detectable by nuclear imaging methods. We discuss crucial tumour properties that can be described by current radioprobe technologies, further discuss areas of emerging radioprobe development, and finally articulate need areas that our field should aspire to develop. The review focuses largely on positron emission tomography and draws upon the seminal ‘Hallmarks of Cancer’ review article by Hanahan and Weinberg in 2011 placing into context the present and future roles of radiotracer imaging in characterizing tumours. Keywords Oncology . Hallmarks . Nuclear imaging . Positron emission tomography . Radiopharmaceuticals Introduction Normal cells have evolved mechanisms for strict homeostatic control of the way in which they acquire biomass (growth) and divide (proliferation). In contrast, not only do tumours deregulate these mechanisms, they also co-opt tumour-associated normal cells within the stroma or tumour microenvironment to support their habit, consequently becoming autonomous. In 2011, Hanahan and Weinberg described the next generation of ‘hallmarks of cancer’ that capture the diversity of neoplastic disease and conceptualize tumour characteristics crucial to their ability to proliferate, invade adjacent tissues and metastasize to distant organs [1]. They expounded the six originally proposed hallmark capabilities – sustaining proliferative I. S. Alam : M. A. Arshad : Q.<D. Nguyen : E. O. Aboagye (*) Comprehensive Cancer Imaging Centre, Imperial College London, London W12 0NN, UK e-mail: eric.aboagye@imperial.ac.uk signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis and activating invasion and metastasis – and added to this two enabling characteristics crucial to the acquisition of the six hallmark capabilities (genome instability/mutations and tumour-promoting inflammation), two new hallmark capabilities (reprogramming tumour energy metabolism and evading immune destruction) and the signalling interactions of the tumour microenvironment (Fig. 1) [1, 2]. It is against this background that we describe radiopharmaceutical probes for characterizing tumours. We discuss crucial tumour characteristics that can be described by current radioprobe technologies, discuss areas of emerging radioprobe development, and finally articulate need areas/ technologies that our field should aspire to develop. Our review focuses largely on positron emission tomography (PET) since this is the most sensitive technology for use in human cancer when all anatomical regions are considered but we also mention a few developments in the analogous single photon emission computed tomography (SPECT) field. Imaging proliferation, growth and replicative immortality Of the hallmark capabilities, the ability to sustain chronic proliferation has perhaps received the most attention in the radiopharmaceutical imaging arena. PET imaging with 18Ffluoro-3′-deoxy-3′-L-fluorothymidine (FLT; Fig. 1), a pyrimidine analogue originally discovered by Shields et al. [3], has been used in several tumour types to characterize cell proliferation [4]. The specific readouts are influenced by the activities of the hENT1 transporter which mediates FLT entry into cells [5] and, perhaps more importantly, thymidine kinase-1 (TK1), a key player in the salvage pathway of DNA synthesis; it phosphorylates the tracer to FLT monophosphate which becomes trapped in cells without 538 Eur J Nucl Med Mol Imaging (2015) 42:537–561 Fig. 1 Overview of tracers for characterizing tumour tissue and the targets for which they are designed to bind or alternatively are activated by. The tracers provide almost complete coverage for highlighting the crucial properties – hallmarks – of cancer. EGFR epidermal growth factor receptor, mutEGFR mutant epidermal growth factor receptor, HER2 human epidermal growth factor receptor-2, ER oestrogen receptor, ENT1 equilibrative nucleoside transporter-1, TK1 thymidine kinase-1, Chk-a choline kinase alpha, AAT amino acid transporter, dCK deoxycytidine kinase, GLUT1 glucose transporter-1, HKII hexokinase-II, CXCR4 chemokine receptor type-4, c-MET protein encoded by the MET gene, VEGF-1 vascular endothelial growth factor-1, CAIX carbonic anhydrase-IX, PARP-1 poly(ADP ribose) polymerase-1, a.m.i. apoptotic membrane imprint, phosphatidyl-ser phosphatidyl serine, ACS acetylcoA synthetase, GS glycogen synthase, FAO fatty acid oxidation, AR androgen receptor, sigma-2 R sigma-2 receptor, SSTR-2 somatostatin receptor-2, TFRC transferrin receptor protein significant incorporation into DNA. Since TK1 protein expression increases as cells enter S-phase and then is targeted for degradation in late M-phase, FLT is exploited as an indirect measure of proliferation [6]. FLT is however unable to detect the de novo synthesis pathway which limits its sensitivity as a proliferation tracer [7]. Uptake can also be influenced by thymidine levels or thymidine phosphorylase expression [8, 9]. The two key canonical suppressors of proliferation – TP53 (or p21) and RB (or RB-E2F complex) – sit upstream of TK1 and consequently regulate its transcription; expression of these proteins can also impact FLT uptake [10]. Other limitations of FLT include its high physiological uptake in liver and bone marrow and relatively low uptake in solid tumours, which currently restricts its use as a diagnostic agent in the clinic [11]. Rather it has shown potential for predicting response to treatment since the majority of chemotherapeutic agents act to inhibit cell proliferation (Table 1) [96, 97]. Radiotracers and selected clinical studies Radiotracer Reference Year Tumour Sustaining proliferative signalling and evading growth suppressors 18 F-FLT [12] 2003 9 NSCLC, 5 oesophageal, 2 sarcoma, 1 Hodgkin’s lymphoma and 1 renal carcinoma 2003 Pulmonary nodules [13] No. of patients Aim and findings of study Design p value 16 Tumour detection. 18F-FLT PET vs. 18F-FDG/Ki-67. Significant difference in SUV values compared to FDG. Bone marrow and liver lesions difficult to detect Tumour detection. 18F-FLT PET vs. 18F-FDG/Ki-67. 18 F-FLT correlated with Ki-67 but was inferior for lesion detection compared with 18F-FDG Tumour detection. 18F-FLT PET vs. clinical, chest radiography, ultrasonography and CT. 88 % sensitivity with pathologically proven lesions Tumour detection. 18F-FLT PET vs. 18F-FDG in 6 patients. No significant difference between 18F-FLT and 18F-FDG and histological markers Tumour detection. 18F-FLT PET vs. 18F-FDG PET correlation with Ki-67. Less sensitive than FDG in detecting primary tumour and no correlation with Ki-67 Tumour detection. 18F-FLT PET vs. 18F-FDG PET correlation with Ki-67. FLT was more sensitive in imaging recurrent high-grade gliomas, correlated better with Ki-67 values, and was a more powerful predictor of tumour progression and OS Tumour detection. 18F-FLT PET vs. 18F-FDG PET. Correlation with Ki-67. Sensitivity in FLT 72 % vs. FDG 89 %. Correlation with Ki-67. Second study: specificity, positive predictive value and accuracy similar to those with FDG Tumour detection. 18F-FLT PET vs. 18F-FDG PET. Correlation with Ki-67. Both tracers had 100 % sensitivity. Higher specificity with FLT. SUVmax of FLT was significantly different between benign and malignant lesions but not that of FDG. Strong correlation with Ki-67 Therapy prediction. Mean change in FLT levels after the first course of chemotherapy correlated with eventual tumour response Therapy prediction. Changes 1-week after chemotherapy correlated with tumour response at 60 days Therapy response. FLT SUVmean decreased significantly by 25 % (p=0.0001) in the absence of significant volumetric change, whereas metastatic nodes decreased in volume by 31 % (p=0.020) with a larger SUVmean decrease of 40 % (p<0.0001) Prospective 0.0006 Prospective NS Prospective NS Prospective 0.25 26 [14] 2003 Melanoma 10 [15] 2004 Breast cancer 12 [16] 2005 Oesophageal cancer 10 [17] 2005 Brain gliomas 25 [18, 19] 2007, 2008 NSCLC 18, 34 [20] 2013 Uterine body tumours 15 [21] 2006 Breast cancer 14 [22] 2007 Breast cancer 13 [23] 2014 NSCLC 16 Eur J Nucl Med Mol Imaging (2015) 42:537–561 Table 1 Prospective Prospective Ki-67 <0.0001, PFS <0.0005, OS <0.001 Prospective <0.0002, second study >0.1 NS <0.01 Prospective <0.01 <0.022 Prospective As stated 539 540 Table 1 (continued) Radiotracer Year Tumour [24] 2011 [25] 2013 [26] 2014 [27] 2014 [28] 2014 11 [29] 2013 18 [30] 2012 [31] 2013 [32] 2014 Lymph node detection. 11C-choline PET vs. MRI and histology (choline kinase alpha and Ki-67). Sensitivity higher for 11C-choline PET/CT compared with MRI (p<0.007). A higher nodal detection rate, including subcentimetre nodes, was seen with 11Ccholine PET/CT than with MRI. Good correlation between SUVmax and choline kinase expression (r=0.63, p<0.0004) Prostate cancer 9 Bone metastasis detection. 11C-Choline vs. bone scan and CT. Increased sensitivity of 11C-choline PET/CT in identifying active lytic lesions and true bony disease Prostate cancer 605 Relapse detection after radical prostatectomy. Comparison with histology and clinical follow-up (with imaging). Detection rate 28 % Prostate cancer 61 Tumour detection in medium-/high-risk patients considered for radical treatment. Extraprostatic disease detected in 24.6 %. Of those with locoregional or oligometastatic disease (73 %), additional radiotherapy was given Prostate cancer lymph node 83 Baseline 11C-choline PET/CT in all vs. change in PSA recurrence (biochemical) and post-radiotherapy 11C-choline (in 47 patients). Biochemical response after treatment was detected in 83 % of patients. The second PET scan detected 17 % (8/47) recurrent disease of which 57 % were at distant sites Prostate cancer Staging 637, Meta-analysis. Staging untreated prostate sensitivity restaging 1,055 84 %, specificity 79 %. Restaging those with biochemical failure after local therapy for curative intent sensitivity 85 %, specificity 88 % Prostate cancer 210 Lymph node detection. 18F-Fluoromethylcholine vs. histology. Sensitivity 56.2 %, specificity 94.0 %. Bone metastases were detected Prostate cancer spinal 50 Bony metastases detection using 18F-FCH PET/CT vs. 18 F-NaF PET/CT and 99mTc-MDP (whole-body metastases bone scintigraphy, WBS) using MRI as reference. Sensitivity: NaF 93 %, FCH 85 %, WBS 51 %. Specificity: NaF 54 %, FCH 91 %, WBS 82 %. Accuracy: NaF 82 %, FCH 95 %, WBS 86 % Prostate cancer 21 Tumour (relapse) detection. 18F-Choline PET/MRI vs. multiparametric MRI (mMRI) vs. 18F-choline PET/ CT vs. 18F-choline PET/CT vs. contrast-enhanced CT (CeCT). 18F-Choline PET/MRI had the highest detection rate (86 %) and accuracy (99 %). 18FCholine PET/MRI higher sensitivity for detecting 11 C-Choline C-Choline and 18Ffluorocholine FFluoromethylcholine 18 F-Choline Prostate cancer No. of patients 28 Aim and findings of study Design p value Prospective <0.007 Prospective NS Retrospective Prospective Prospective NS Retrospective NS Prospective Prospective Prospective Eur J Nucl Med Mol Imaging (2015) 42:537–561 Reference Radiotracer 18 F-D4-choline Zr-Cetuximab 89 18 F-FES 89 Zr-Trastuzumab Invasion and metastases 18 F-Sodium fluoride (NaF) Angiogenesis 89 Zr-Bevacizumab 15 O-Water C-Carbon monoxide Year Tumour No. of patients [33] [34] 2014 2013 Normal volunteers Head and neck 8 268 [35] 2011 Endometrial carcinoma 19 [36] 2013 Uterine 47 [37] 2010 Breast cancer 14 [38] 2008 Prostate cancer metastases 38 [39] 2011 Breast, prostate 34 [40] 2013 Breast cancer 23 [41] 2009 14 [42] 2012 Synovial sarcoma, Breast cancer, malignant melanoma, neck adenocarcinoma, colon cancer, oropharyngeal SCC, leiomyosarcoma, liposarcoma, chondrosarcoma Renal, colon, NSCLC, oesophageal [43] 2003 Prostate cancer 6 7 Aim and findings of study lymph node metastases than mMRI (p=0.0002) and CeCT (p<0.0001) Biodistribution Multicentre trial. Detection and treatment response. Ongoing Tumour detection. 18F-FES vs. 18F-FDG PET. FES uptake significantly positively correlated with expression of oestrogen receptor α (ERα). The FDG to FES ratio significantly negatively correlated with expression of ERα and progesterone receptor B. FDG uptake not correlated with any of the immunohistochemical scores 18 F-FES and 18F-FDG PET uptake correlated with expressions of sex hormone receptors, GLUT-1 and Ki-67 in mesenchymal uterine tumours. 18FFDG/18F-FES ratio correlated with Ki-67, GLUT-1 and ERb in uterine sarcoma Biodistribution and tumour detection Design p value Prospective Prospective NS Prospective Prospective NS Prospective Bone metastasis detection. 18F-NaF vs. 18F-FCH PET. Prospective With 18F-NaF sensitivity higher (81 % vs. 74 %, p=0.12), and specificity lower (93 % vs. 99 %, p=0.01) NS Bone metastasis detection. 18F-NaF vs. bone scan scintigraphy. With 18F-NaF higher sensitivity (76 vs. 45 %), specificity (84 vs. 79 %) and accuracy (80 vs. 60 %) Prospective Tumour detection. Of 26 tumours, 25 were detected and there was a correlation with VEGF-A in the histology sample (r=0.49) Tumour detection. Good correlation between perfusion Retrospective CT and 15O-water Treatment response with sunitinib therapy. Baseline Prospective and posttreatment scans vs. 18F-FDG PET. Significant change in levels in responders (p<0.001) compared with nonresponders (p=0.38) Prospective 0.004 <0.001 541 11 Reference Eur J Nucl Med Mol Imaging (2015) 42:537–561 Table 1 (continued) 542 Table 1 (continued) Radiotracer 18 F-Galacto-RGD Reference Year Tumour No. of patients 2007 Head and neck SCC [45] 2008 [46] 2011 NSCLC, rectal 18 adenocarcinoma, renal cell carcinoma, head and neck cancer, breast cancer, haemangiosarcoma, carcinoid of bronchus Breast cancer 6 F-RGD-K5 [47] Genome instability and mutation 124 I-cG250 [48] 2012 Healthy volunteers 4 2005 Renal cell carcinoma 20 [49] 2013 Renal cell carcinoma 195 [50] 2006 NSCLC 8 [51] 2011 53 [52] 2013 Metastatic renal cell carcinoma Pancreatic cancer [53] 2013 18 F-Fluciclatide 18 18 F-FMISO Head and neck, gastrointestinal, uterine, lung cancer 11 10 10 Design Treatment response using 11C-carbon monoxide vs. 15 O-water, 18F-FDG PET and PSA. Moderate correlation in 11C-carbon monoxide with change in PSA (r=0.65, p=0.14) and percent change tumour blood volume and SUVmax (r=0.60, p=0.19). Strong correlation between percent change in tumour blood volume and SUVmean (r=0.82, p=0.032) Tumour detection. Correlation with Prospective immunohistochemical avh3 expression Tumour detection and comparison with 18F-FDG PET. Prospective Sensitivity compared to clinical staging 76 %. SUVs significantly higher with FDG (p<0.001). No correlation between the two radiotracers (r=0.157; p=0.235) Tumour detection. Higher binding in lung metastases compared with normal lung and lower binding in liver metastases compared with normal liver Biodistribution Tumour detection. Comparison of 124I-cG250 vs. 18FFDG PET. 18F-FDG detected more metastases, 69 % vs. 30 %, than 124I-cG250 Tumour detection. Comparison of 124I-cG250 with contrast-enhanced CT. Sensitivity higher with 124IcG250 (86 % vs. 75.5 %, p=0.023) and specificity (85.9 % vs. 46.8 %, p=0.005) Therapy response. 18F FMISO vs. 18F-FDG PET before and after two cycles chemotherapy. No association between tumour hypoxia detected by FMISO PET and tumour glucose use measured by FDG PET. Some discrepant findings between the two radiotracers due to distant metastases. FDG SUV decrease associated with response Therapy response. Follow-up clinical and with CT. Changes in tumour hypoxia not related to PFS or OS Tumour detection. 18F FMISO PET vs. 18F-FDG PET. No relationship between 18F-FDG or 18F-FMISO activity and histological grade or radiological staging Therapy response. Before and after chemoradiotherapy scans demonstrated that those that tumours with reduced hypoxic volumes after treatment p value >0.1 >0.05 Prospective Prospective Prospective Prospective Prospective NS Prospective PFS 0.16, OS 0.82 Prospective SUVmax 0.41 Prospective NS Eur J Nucl Med Mol Imaging (2015) 42:537–561 [44] Aim and findings of study Radiotracer Reference Year Tumour No. of patients 64 [54] 2014 Head and neck 11 18 [55] 2011 Prostate cancer 14 [56] 2013 NSCLC 11 [57] 2013 Rectal cancer 14 [58] 2013 NSCLC 11 [59] 2008 Breast cancer 16 [60] 2012 Head and neck 12 [61] 2013 NSCLC 15 [62] [63] 2014 2014 Healthy volunteers NSCLC, head and neck cancer 4 1,019 [64] 2002 Lymphoma, lung cancer, breast cancer 15 Cu-ATSM F-Fluoroazomycin arabinoside (FAZA) 18 F-HX4 18 F-VM4-037 Texture analysis and radiomics Cell death 99m Tc-Annexin V Aim and findings of study corresponded with partial or complete response with long-term local control Therapy response. 64Cu-ATSM vs. 18F-FDG PET before and after treatment. The 64Cu-ATSM scans showed high sensitivity (100 %) but low specificity (50 %) in predicting neoadjuvant chemoradiotherapy response. No difference between the biological tumour volumes between the two radiotracers Tumour detection. 18F-FAZA vs. MRI/histology. No correlation found between 18F-FAZA uptake and Gleason scores Tumour detection. 18F-FAZA vs. 18F-FDG PET. 18FFAZA able to detect heterogeneous distributions of hypoxic subvolumes out of homogeneous 18F-FDG background Tumour detection. 18F-FAZA PET had higher SUVmax and SUVmean compared to background muscle and bowel Therapy response. 18F-FAZA PET vs. 18F-FDG PET. Intralesional hypoxia detected in 65 % of pretreatment scans, which resolved after treatment. Disease-free survival not significantly different between hypoxic and non-hypoxic groups Tumour detection. 18F-Galacto-RGD detected all primary tumours but only three out of eight lymph node metastases Tumour detection. Comparison with 18F-FMISO and 18 F-FDG (ten patients) vs. surgery (nine). Higher sensitivity with 18F-HX4 vs. 18F-FMISO (100 % vs. 50 %) and specificity (85.7 % vs. 71.4 %). Strong correlation between the two tracers (r=0.84) Tumour detection. Comparison between acquisition at 2 h and 4 h after injection with no significant difference in the heterogeneous pattern uptake between the two time-points Biodistribution Four textural features from the test set had high concordance on the validation set with underlying gene expression and prognosis Design p value Prospective NA Prospective Prospective Prospective <0.004 Prospective 0.42 Prospective NS Prospective <0.05 Eur J Nucl Med Mol Imaging (2015) 42:537–561 Table 1 (continued) Prospective Prospective Prospective and retrospective <0.05 543 Therapy response. Increased annexin uptake 24–48 h Prospective after chemotherapy related to complete response/ partial response. No uptake related to stable or progressive disease. OS and PFS were significantly <0.05 on different datasets 544 Table 1 (continued) Radiotracer Reference Year Tumour No. of patients Aim and findings of study [65] 2004 Follicular lymphoma 11 99m [66] 2004 NSCLC, lymphoma, leukaemia, head and neck SCC 33 99m [67] Head and neck SCC 13 18 [68] 2012 Brain metastases 10 18 [69] 2013 Healthy volunteers 8 F-CP-18 [70] Deregulating cellular energetics 11 C-Acetate [71] 2013 Healthy volunteers 7 Biodistribution 2012 Urothelial cancer 14 [72] 2013 Hepatocellular carcinoma 43 [73] 2013 Prostate cancer 46 [74] 1994 Lung cancer 23 [75] 1996 Head and neck 30 Tumour detection. 11C-acetate PET vs. 11C-choline PET and histology. No significant difference between the radiotracers in detecting tumours, lymph nodes or prostate pathology Tumour detection. 11C-acetate vs. 18F-FDG PET/ contrast-enhanced CT/histology. The overall sensitivity (96.8 %) and specificity (91.7 %) of dual-tracer PET/CT for patient selection for liver transplant were significantly higher than those of contrast-enhanced CT (41.9 % and 33.0 %, respectively; both p<0.05). Overall staging accuracy for 11C-acetate was 90.5 % vs. 33.3 % for FDG Tumour detection. 11C-acetate PET/CT vs. 18F-FDG PET and CT/bone scintigraphy/biopsy. 11C-acetate detected 30 % of disease compared with 9 % with FDG PET Tumour detection. 18F-FDG vs. CT. PET and PET fusion images had higher sensitivity (82 % vs. 64 %), specificity (82 % vs. 44 %) and accuracy (81 % vs. 52 %) Tumour detection. Fused FDG PET/CT and FDG PET MRI vs. CT/MRI. Higher accuracy with fused PET Tc-Annexin V Tc-HYNIC-annexin V Tc-HYNIC-annexin V F-ML-10 F-ICMT-11 18 18 F-FDG p value Prospective NS Prospective NS Prospective NS Prospective NS Prospective 0.06 Retrospective NS Prospective NS Prospective <0.05 Prospective Eur J Nucl Med Mol Imaging (2015) 42:537–561 99m related to tracer uptake in treated lung cancers and lymphomas Therapy response. Uptake increased after 4 Gy – correlated with clinical outcome and cytology Therapy response. Increased uptake associated with complete/partial response after radiotherapy/ chemotherapy. Suggested that annexin V may be used as a predictive marker for early treatment response Therapy response. Glands that received higher doses showed more annexin uptake. No correlation between uptake and patient outcome. Baseline necrosis in tumours a confounding factor Therapy response. Baseline and follow-up 18F-ML-10 compared with MRI after radiotherapy. Highly significant correlation between early changes on the 18 F-ML-10 scan and later changes in tumour anatomical dimensions (r=0.9) Biodistribution Design Radiotracer Reference Year Tumour No. of patients [76] 1997 Lymphoma 18 [77] 1998 Head and neck 24 [78] 1998 Oesophageal 26 [79] 2000 Non-Hodgkin’s lymphoma 49 Prostate cancer 7 [81] 2014 Prostate cancer 38 18 [82] 2013 Lymphoma, breast cancer, head and neck cancer 30 68 [83] 2007 Neuroendocrine tumours 84 [84] 2008 Neuroendocrine tumours 38 [85] 2014 Neuroendocrine tumours 416 F-ISO-1 Ga-Somatostatin receptors (SSRs) images (98 % vs. 69/40 %). Management altered in 7/30 patients Tumour detection. FDG PET compared with wholebody CT. Results compared with histology and clinical follow-up. Both methods detected exactly the same number of lesions but FDG PET was more cost-effective Lymph-node detection. Comparison vs. CT and MRI. Sensitivity (87.5 % vs. 53.1 %), specificity (99 % vs. 87.8 %) and accuracy (98.2 % vs. 85.3) higher with PET. Negative predictive value 99 % Tumour detection. FDG PET vs. CT in comparison with histology. Higher primary tumour detection (96 % vs. 81 %) and lymph node detection (90 % vs. 62 %). Diagnostic accuracy for surgical resectability was 88 % vs. 65 %, respectively, and PET and CT combined 92 % Tumour detection. Comparison with CT. FDG PET able to predict disease outcome (p<0.001). Interim PET was able to provide long-term prognosis (p<0.01) Tumour detection. 18F-FDHT vs. 18F-FDG PET. 18FFDG PET positive in 57 of 59 lesions (97 %). 18FFDHT PET positive in 46 of 59 lesions (78 %). Treatment with testosterone resulted in diminished 18 F-FDHT uptake at the tumour site Bone metastasis detection. 18F-FDHT vs. 18F-FDG and CT. Lesions on all modalities significantly associated with OS. intensity of the FDHT also associated with OS (p=0.02) Tumour detection. 18F-ISO-1 vs. Ki-67. Tumour SUVmax and tumour-to-muscle ratio correlated significantly with Ki-67 (τ=0.27, p=0.04, and τ=0.38, p=0.003, respectively) Tumour detection. 68Ga-DOTATOC PET vs. 99mTcHYNIC-TOC and 111In-DOTA-TOC. 97 % sensitivity and 92 % specificity with accuracy of 96 % for PET Tumour detection. 18F-DOTATOC PET vs. 18F-FDG PET, Ki-67. Sensitivity higher with DOTATOC (82 %) than with FDG (62 %). Combined sensitivity (92 %), correlation with tumour grade (p<0.001) Design p value Prospective Prospective Prospective Retrospective Prospective Retrospective 0.02 Prospective Prospective <0.001 Retrospective 0.005 Retrospective NA 545 Specific cell antigens, receptors and transporters 18 F-FDHT [80] 2004 Aim and findings of study Eur J Nucl Med Mol Imaging (2015) 42:537–561 Table 1 (continued) 546 Table 1 (continued) Radiotracer 18 F-DOPA Reference Year Tumour No. of patients 2001 Gastrointestinal carcinoid 17 [87] 2007 Gastroenteropancreatic tumours 84 [88] 2009 Medullary thyroid carcinoma 26 [89] 2012 Glioblastoma 32 [90] 2012 Prostate cancer 20 [91] 2012 Gliomas 102 [92] 2013 Intramedullary tumours 9 18 F-FACBC [93] 2014 Prostate cancer 28 11 C-Metomidate [94] 2006 Adrenocortical tumours 73 [95] 2013 Liver tumours 33 11 C-Methionine Meta-analysis comparing 68Ga-DOTATOC PET vs. 68 Ga-DOTATATE PET. Both had high sensitivity (93 % vs. 96 %), and specificity (85 % vs. 100 %) Tumour detection. 18F-DOPA PET vs. 18F-FDG PET/ SSR scintigraphy/CT or MRI. Sensitivities: 18FDOPA PET 65 %, 18F-FDG PET 29 %, SSR scintigraphy 57 %, morphological procedures 73 % Tumour detection. 18F-DOPA vs. ultrasound/CT/MRI and 111In-pentetreotide scintigraphy. 18F-DOPA PET/CT detected the primary tumour in all cases where the other imaging modalities failed and detected a further 12 unsuspected lesions. Clinical management changed in 11/13 pts (84 %) 18 F-DOPA PET/CT vs. 18F-FDG PET/CT. DOPA PET correctly detected 94 % of malignant lesions (50/ 53), but FDG PET detected only 62 % (33/53) Tumour detection. 11C-Methionine PET/CT vs. CT, Gd-enhanced T1-W and T2-W MRI. 11CMethionine as gold standard Tumour detection. 11C-Methionine PET/CT vs. 18FFDG PET and histology. No significant difference between the two radiotracers in ability to diagnose prostate cancer with Gleason score >8. MET appears useful for detecting prostate cancer of both low and high Gleason score Tumour detection. 11C-Methionine vs. 18F-FDG PET/ contrast-enhanced MRI. 11C-MET PET predicted prognosis in gliomas and better than 18F-FDG PET and MRI in predicting survival in low-grade gliomas Tumour detection. 11C-Methionine vs. 18F-FDG PET/ MRI. Higher SUVmax with FDG than with methionine. FDG accumulates in haemorrhage Tumour detection. 18F-FACBC PET vs. 11C-choline. The detection rate with anti-3-18F-FACBC PET/CT greater than with 11C-choline, with approximately 20 % of additional patients and approximately 60 % additional lesions detected Tumour detection. 11C-Metomidate vs. histology. Sensitivity 89 % and specificity 96 % in proving adrenocortical origin of tumours. All metastases and non-adrenal lesions were negative Tumour detection. 11C-Metomidate vs. 11C-acetate/ MRI. Low sensitivity of both radiotracers NS not stated, SCC squamous cell carcinoma, NSCLC non-small-cell lung cancer, PFS progression-free survival, OS overall survival Design p value Prospective Prospective Prospective Prospective NA Prospective Retrospective 0.04 Prospective NS Prospective NS Retrospective NS Prospective Eur J Nucl Med Mol Imaging (2015) 42:537–561 [86] Aim and findings of study Eur J Nucl Med Mol Imaging (2015) 42:537–561 Imaging probes also exist for cell surface receptors that initiate mitogenic signalling including epidermal growth factor receptor (EGFR), HER2 and the oestrogen receptor (ER). Numerous imaging strategies for EGFR detection have been developed based on radiolabelling of EGFR-specific monoclonal antibodies such as cetuximab, Affibodies, nanobodies and small-molecule inhibitors such as erlotinib [98]. Using the 4-(anilino)quinazoline derivative, [11C]PD153035, an attempt was made to characterize EGFR tyrosine kinase expression in patients with non-small-cell lung cancer (NSCLC) [99]. Patients with higher tumour radiotracer uptake had higher overall and progression-free survival (overall survival 11.4 vs. 4.6 months, p=0.002). Regarding radiolabelled cetuximab, the advantage of using a directly radiolabelled clinically available IgG (reduced regulatory burden) should be balanced against the slow pharmacokinetics of 89Zr-cetuximab. As recently demonstrated, new methods for pretargeting involving a bioorthogonal inverse electron-demand Diels-Alder reaction may allow the use of an appropriately radiolabelled tetrazine in conjunction with the IgG, following clearance from the circulation, to improve contrast and reduce dose to subjects [100]. Recently possibilities for detecting activating mutations of EGFR have also been outlined [101–103]. The presence of activating mutations in the kinase domain of the EGFR receptor increases response rates from <10 % in patients with wildtype EGFR to >80 % in patients with the activating mutations. 18 F-PEG6-IPQA developed by the Gelovani Laboratory, shows higher affinity for L858R active mutant EGFR in comparison with wild-type EGFR, where the presence of the former is associated with favourable responses to therapy. The increased affinity for the active mutant has been demonstrated in lung carcinoma cells in vitro and using an in vivo NSCLC model [101, 103]. This pegylated quinazoline tracer is currently being considered for clinical use in the selection of NSCLC patients who would show favourable responses to smallmolecule EGFR kinase inhibitors with a similar structure (Clinicaltrials.gov NCT01320059). Another member of the EGF family of receptors, HER2, is an important prognostic and predictive factor in breast cancer, with approximately 20 % of patients expressing the receptor. Several probes for this target have been developed. These include clinically approved IgG molecules (about 150 kDa) such as 89Zr-trastuzumab [37, 104], antibody fragments including 68Ga-DOTA-HER2 F(ab’)2-trastuzumab [105], and much smaller Affibodies (about 6.5 kDa) including 18F-GE226 for PET [106] and 111In-ABY-025 for analogous single photon imaging [107]. The long time to achieve contrast (4– 5 days) due to the slow pharmacokinetics of IgGs and a requirement for titrating the optimal dose of the tracer in drugnaive patients are limitations of the antibody approach. While the Affibodies have theoretical advantages over IgGs, clinical trials to assess their full potential need to be completed. Whichever probe eventually evolves as the clinically routine 547 probe for HER2 imaging may find utility, particularly in the metastatic setting, for stratifying patients receiving HER2directed therapies. The labelled probes mentioned above have already been used to characterize tumour receptor expression in multiple anatomic sites, and also the effect of drugs that degrade the HER2 including HSP90 inhibitors. PET imaging of ER is also a diagnostic tool in breast cancer patients. The ER-specific tracer 18F-FES has been used to characterize breast and uterine tumours with high sensitivity for noninvasive reporting of ER status [35, 36, 108]. Tsujikawa et al. showed that 18F-FES uptake in breast cancer patients is positively correlated with expression of ERα while 18 F-FDG uptake as expected failed to correlate with immunohistochemical scoring [35]. With the exception of liver metastasis in which physiological uptake of the tracer is already high, 18F-FES shows good sensitivity for detection of metastatic lesions [108]. An important finding from 18F-FES PET in characterizing tumours is the confirmation that patients with no receptor expression in their metastasis, despite previous detection of receptor positivity in their primary tumours, have a low likelihood of responding to endocrine therapy [109]. In contrast to the imaging of cell surface receptor factors, radioprobes for branched downstream pathways including PI3K and ERK are still lacking. It is currently not possible to directly trace within specific cancers expression of crucial somatic mutations that activate signalling, disrupt negative feedback or evade tumour suppression, e.g. BRAF V600E, PTEN, or protein products of tumour suppressor genes that affect contact inhibition, e.g. NF2 and LKB1. An area that has recently received some attention is signalling factors in the context of quiescence (and by extension, senescence). In lymphoma models of cancer, Dorr et al. found that therapyinduced senescence that is also characterized by expression of the S-phase entry-blocking histone-3 lysine-9 trimethylation mark is described as a negative change on 18 F-FLT PET accompanied by a positive change on 18FFDG PET [110]. Whether this phenotype is limited to this particular model of senescence or more broadly applicable remains to be seen. The laboratory of Aboagye recently reported a novel radioprobe for imaging glycogenesis described later [111]. The probe could also find utility in rationalizing the ability of cancer cells presenting with excessive oncoproteins, e.g. RAS oncogene family member RAB25, to activate quiescence/senescence or survive under bioenergetic stress conditions. There are certain proliferation-linked hallmark capabilities, including sustenance of replicative immortality, that have not been directly evaluated for imaging. It is thought that imaging of proliferation indirectly infers de facto replicative immortality. A key feature of this trait is the ability of cancer cells to counter telomeric DNA erosion by expressing the enzyme telomerase, and in doing so to achieve immortality [112, 113]. While telomerase per se could be targeted for imaging, 548 this activity has not been reported in the field of PET. Liu et al. have reported a SPECT probe for detecting human telomerase reverse transcriptase (hTERT), the major component of telomerase activity, using antisense oligonucleotides (99mTchTERT ASON) [114]. Though higher tracer accumulation was observed in vivo in MCF7 xenografts compared to labelled control sense oligonucleotides, image quality was compromised due to high background. Cancer stem cells (CSCs) present in the tumour microenvironment are known to show the ability for self-renewal and have been suggested to be responsible for long-term tumour propagation [115]. Thus, identification of CSCs has received much attention because of their potential for therapeutic targeting [116]. Although Hanahan and Weinberg [1] discuss the role of stem cells in the tumour microenvironment, we consider here that CSCs also contribute to replicative immortality (Fig. 1). CD133 is the most prominent and widely used biomarker in CSC research. Recently, the Weber laboratory reported a new radioprobe for imaging CD133 based on detection of a specific glycosylation-dependent epitope called AC133 using a 64Cu-labelled AC133-specific monoclonal antibody [117]. The tracer shows high stability in vivo, with impressive retention in AC133+ tumours both in superficial xenograft and orthotopic models of glioblastoma. While this represents a milestone achievement in the field, there is some hesitation about the specificity of detecting CSCs using this particular method. The expression pattern of CD133, though widely associated with CSCs, is not specific to this population of cells and is also associated with epithelial and nonepithelial cells [118]. Certain treatments are known to influence glycosylation of the antigen and thus the ability of the antibody to bind. Additionally some treatments interfere with the stability and expression of the entire CD133 antigen on the cell surface [118, 119]. To date there is no single receptor that can be used exclusively to identify CSCs in various cancers. Targeting of CD133 along with additional CSC-associated receptors such as CD44 may yield enhanced specificity [120]. Imaging cell death Another area that has received significant interest in radioprobe development is the characterization of cell death, in particular programmed cell death or apoptosis in which the onset and extent of cell death after treatment is considered a positive prognostic indicator. Both ‘extrinsic’ and ‘intrinsic’ circuits of apoptosis are important in activating a set of proteases – caspases – to execute the apoptosis programme. Activation of specific caspases, e.g. caspase 3 and caspase 7 from latent procaspases, can be detected by PET imaging. The isatin-5-sulphonamide family having subnanomolar affinity for caspase 3 and 7 has been developed to image this committal stage of apoptosis. ICMT-11 was chosen because of its Eur J Nucl Med Mol Imaging (2015) 42:537–561 high affinity for activated caspase 3, reduced lipophilicity and easy radiolabelling. Preclinical studies with the tracer in murine lymphoma xenografts, and breast and colon models are promising though the transient nature of the target makes it challenging to establish the best time to image [121, 122]. Additionally because of its lipophilicity, the tracer exhibits hepatobiliary clearance with high uptake observed in the liver, gall bladder and small intestine. 18F-ICMT-11 is currently in clinical trials for imaging caspase activation in patients and so far has shown favourable dosimetry in healthy subjects [69, 123]. In the arena of cell death, phosphatidylserine (PS) externalization closely follows caspase activation, and changes in cellular and mitochondrial membrane potential are other generic features of apoptosis. These are amenable to tracing by SPECT and PET with annexin V-based imaging probes which have high affinity for PS and by PET with ML-10 (5fluoropentyl-2-methyl malonic acid) to detect a combination of these factors (Fig. 1) [68, 124]. Annexin V has been derivatized with a variety of SPECT radioisotopes and chelates since its characterization in the mid-1990s but from a clinical standpoint and despite reaching phase II/III clinical trials, has been plagued by poor biodistribution with high uptake in the liver and kidneys which precludes imaging in the abdominal region (Table 1) [64, 125, 126]. The probe is also unable to detect dying cells when accessibility is compromised because of disruption to vasculature in the context of antiangiogenic therapy [127]. Another similar but smaller PS binding protein based on the C2A domain of synaptotagmin I has been shown to be more specific than annexin V for dying cells in vitro [128]. It has yet to reach the clinic but has shown promising results in preclinical models with a more favourable clearance profile than annexin Vowing to its smaller size. 18FLabelled versions of annexin V [129, 130] and C2A [131] have also been developed for PET. The commonality between the two PS binding proteins is that they ultimately lack specificity for apoptosis and show retention in necrotic and inflamed tissue as well as platelets. ML-10, from the Aposense family of small molecules also has a more favourable biodistribution and clearance profile than annexin V due its low molecular weight, as well as acceptable dosimetry and stability demonstrated in first inhuman studies [132]. Based on this, the tracer has been used to image apoptosis in brain tumours in patients and has shown high tumour uptake. Indeed early radiotherapy-induced changes shown by 18F-ML-10 correlate well with late changes in tumour size as confirmed by MRI (Table 1) [68]. The precise mechanism of accumulation of this tracer still requires clarification. In addition to these broad generic mechanisms, specific targets along the branched pathways of cell death may be altered by tumour cells to resist apoptosis. In particular, the cellular sensors of apoptosis, e.g. TP53 (in response to DNA Eur J Nucl Med Mol Imaging (2015) 42:537–561 damage), may be lost, intracellular apoptosis triggering proteins Bax or Bim may be downregulated and antiapoptotic proteins Bcl2 or Bcl-xL may be overexpressed. To date radioprobe strategies to interrogate these more specific factors have not been developed, in part due to their cell lineage specificity, as well as the shear multiplicity of factors necessitating multiplexing of several radioprobes to provide readout of apoptosis resistance, which is impractical. Equally radioprobes have yet to be developed to specifically measure processes involved in necrosis or autophagy – a survival mechanism that is invoked by cancer cells under various physiologically stressful conditions or following treatment with pharmacological inhibitors and radiotherapy. Apart from its cytoprotective role which could lead to persistence of tumour cells, autophagy has also been implicated in prevention of tumour development in the early stages of tumorigenesis [133]. The role of autophagy in therapeutic response still requires significant clarification to motivate development of radioprobes in this area. Imaging tumour angiogenesis The last two hallmark capabilities involve an interaction between the cancer cell and its host. The ability of cancers to increase biomass is linked to an efficient delivery of nutrients and elimination of byproducts. Angiogenesis – the development of new blood vessels – is transient in normal cells but ‘switched-on’ in tumours [134]. Again a myriad of factors exist to activate or inhibit angiogenesis, and these factors together with the phenotype they provoke, can be exploited for imaging using radioprobes. Imaging of angiogenesis growth factors and interaction with their cognate receptors, e.g. VEGF and VEGFR, has been undertaken preclinically with 64CuDOTA-VEGF and clinically with 89 Zr-bevacizumab immunoPET [40] (Table 1). However, most activity in the field relates to detection of αvβ3 and αvβ5 integrin receptors, factors overexpressed in tumour vasculature compared to mature vessels. RGD (arginine-glycine-aspartic acid) peptides are able to bind αvβ3/αvβ5 with high affinity and RGDbased tracers such as 18F-galactoRGD and 18F-fluciclatide have been used in patients (Table 1) [46, 135]. 18 FGalactoRGD, developed by Beer and co-workers, features a sugar moiety introduced to enhance clearance and has shown promising results in head and neck cancer [44], breast cancer [59] and other malignancies [45]. Its complicated multistep synthesis fuelled the development of a second-generation compound, 68Ga-NODAGA, which has shown promising results in early preclinical studies [136]. In comparison to 18FgalactoRGD, 18F-fluciclatide can be more easily synthesized and shows suitable biodistribution and stability in healthy volunteers. A phase I study in seven patients with metastatic breast cancer showed good tumour to nontumour ratios whilst 549 metastatic lesions in the liver manifested as hypointense regions with lower uptake than healthy liver [137]. The tracer ideally requires further clinical characterization in other types of oncological disease. In the breast cancer study by Kenny et al., several lesions were depicted as ‘doughnut-shaped’ masses confirming the known anatomical appearance of the angiogenesis phenotype. The above studies complement haemodynamic measurements of tumour perfusion detected by 15O-H2O dynamic PET imaging, considered the gold standard for imaging tumour blood flow in patients [138]. 15O-H2O has been applied successfully in clinical studies to evaluate antiangiogenic therapy [139]. Because angiogenesis is necessary for expansion of nearly all tumours and occurs early in cancer development, it has become an important area of research aiming to utilize the radioprobes targeting angiogenesis as generic diagnostic imaging tools. Of relevance to tumour characterization, some tumours, e.g. pancreatic tumours with their characteristic high stromal content, can be very hypovascular, while others e.g. renal tumours, can be hypervascular, providing a cautionary note for the broad exploitation of this phenotype for diagnostic purposes. The biological relevance of detecting this phenotype is that it can aid elucidation of mechanisms of tumour angiogenesis/perfusion or conversely its inhibition for therapeutic benefit. Another important characteristic of tumours, in the context of anti-angiogenesis therapy, is the ability to normalize their vasculature and associated interstitial pressure [140, 141]. Again multiplexing radioprobes with other modalities including MRI can permit such biology to be appreciated in human tumours [142]. Imaging tumour invasion and metastasis In the majority of cancers, it is the invasion and metastasis of tumours that leads to the demise of the patient, yet there is a limited number of specific radioprobes to characterize these processes. Whereas characterizing the processes of cellular invasion may support strategies to reduce/prevent metastasis (depending on whether one views metastasis as an early event of dissemination and dormancy prior to colonization or as a late event in tumour growth), characterizing metastasis may also offer ways of targeting distinct aggressive phenotypes. The processes of local invasion, intravasation of cells into blood and lymphatic systems whether through amoeboid or other forms, extravasation into distant organs and colonization of those organs [143] are perhaps too difficult to visualize in living subjects using radioprobes and such investigations are usually the preserve of high-resolution microscopy [144]. There are nonetheless a myriad of processes that could be targeted for nuclear imaging to investigate various metastatic phenotypes including loss of E-cadherin involved in cell-tocell adhesion and maintenance of epithelial cell sheets, 550 upregulation of other adhesion molecules that promote cell migration such as N-cadherin, epithelial–mesenchymal transition and cancer–stromal cell interactome. Of these the cancer– stromal cell interactome – specifically the interaction between CXCR4 expressed on tumour cells and its cognate ligand CXCL12 expressed on tumour-associated stromal cells - has received the most attention in the imaging field (Fig. 1). Radioprobes of diverse chemical composition, including tetradecapeptides and cyclopentapeptides, and small molecules, have been developed as agents for investigating CXCR4 expression. The most prominent group of peptidederived CXCR4 imaging agents are based on a 14 amino acid peptide sequence known as T140. T140 derivatives such as Ac-TZ14011, have high affinity in the low nanomolar region for CXCR4, as well as high specificity. Modification of AcTZ14011 with bifunctional chelate for incorporation of the SPECT isotope 111In resulted in a decrease in affinity for CXCR4 of almost sixfold. Despite reduced affinity, tumour tissue was clearly visualized, although the extent of uptake in the liver and spleen rendered the tracer unattractive for clinical development [145]. The T140 pharmacophore subsequently underwent further development to generate smaller cyclopentapeptides such as FC131 with similar binding affinity, although here too, high liver uptake represents a constant feature. Only one tracer for PET, acyclopentapeptide ([68Ga]CPCR4.2 [146]), has completed initial evaluation in humans. Among small-molecule radioprobes, those of the cyclam family have received considerable attention as CXCR4 inhibitors. Radiolabelled AMD3100 has shown considerable uptake in the liver and lymphoid organs, which was mostly specific in nature as demonstrated later in blocking studies, and can be explained by the expression of the target in the liver on leucocytes and monocytes [147, 148]. Phase 0 human studies of 64Cu-AMD3100, originally developed by Nimmagadda et al. [147], have commenced (Clinicaltrials.gov NCT02069080). Further developments yielded AMD3465, a monocyclam, with higher affinity for CXCR4 and smaller size and charge than earlier cyclams. When labelled with 64Cu, AMD3465 shows superior target specificity within this family of molecules, good pharmacokinetics and high tumour uptake, but also high liver retention which is the main obstacle in its clinical development [149]. Current research in this field is focused on generating the optimal radioprobe. It is envisaged that the next important stage in development will be the characterization of the invasive potential and resistance to therapy of tumours using the optimal radioprobe. The overexpression of c-MET tyrosine kinase proto-oncogene, a receptor that binds the ligand hepatocyte growth factor, is also implicated in cancer cell invasion, and a number of anti c-MET therapies are in clinical development [150]. PET imaging of c-MET overexpression has been performed at the preclinical level using the peptide 18F-AH113804, which has high affinity for human c-MET receptor and was able to Eur J Nucl Med Mol Imaging (2015) 42:537–561 distinguish among different levels of c-MET expression in three distinct tumour models [151]. Additionally, the peptide has been used in nonhuman primates with high specific binding in the liver and a favourable clearance profile [152]. Again, its use in characterizing clinical tumour aggressiveness remains to be explored. Finally, the downstream osteogenic consequences of metastasis in bone are also detectable by PET. 18F-NaF binds to calcium ions in hydroxyapatite crystals of bone and has been used in the clinic to detect bone metastases. 18F-NaF PET has been shown to detect lesions with higher sensitivity than 18FFCH in prostate cancer metastases [38]. In a separate study involving patients with breast and prostate metastases, 18FNaF PET has also shown higher specificity than bone scintigraphy using 99mTc-labelled methylene diphosphonate (MDP) owing to its superior pharmacokinetics that result in higher bone uptake and faster clearance (Table 1) [39]. Imaging genome instability in cancer Underlying the functional capabilities described above as the hallmarks of cancer is the accumulation of alterations in the genome of neoplastic cells that lead to instability. Described as an enabling characteristic, genome instability involves the acquisition of mutations through a multitude of mechanisms including compromised surveillance systems. These mutations confer a selective advantage enabling cells harbouring them to dominate in the local tissue environment. Much of the nuclear imaging activities probing for accelerators of this instability have focused on hypoxia, a key driver of genome instability [153]. Hypoxia – reduced tissue oxygenation – is known to directly affect gene expression in leading to suppression of apoptosis and promotion of hallmarks such as angiogenesis and invasion but also genomic instability [154]. In solid tumours hypoxia is associated with resistance to radiotherapy and chemotherapy and aggressive tumour types, thus tracer development in this area has been explored as a strategy to predict treatment outcome [155]. The most widely studied radiotracer in this group is 18F-fluoromisonidazole (18F-MISO), a nitroimidazole which becomes reduced and trapped within hypoxic cells (Fig. 1). A number of clinical studies using 18F-MISO have supported the validity of baseline hypoxia assessment with the tracer as a means of predicting treatment outcomes (Table 1) [51, 156]. Limitations reported for this tracer include relatively slow clearance and low tissue uptake which lead to poor contrast. Second-generation 2-nitroimidazoles were subsequently developed to be more hydrophilic and thus have superior biokinetic characteristics, including faster clearance. These include 18F-FETNIM [157], 18F-FETA [158], 18F-HX4 [159, 160] and 18 F-FAZA [161, 162]. Indeed, these secondgeneration probes have shown superior metabolic stability Eur J Nucl Med Mol Imaging (2015) 42:537–561 and clearance profiles and thus sensitivity in preclinical and clinical evaluation (Table 1). 18F-HX4 represents a new click chemistry-based generation of tracer in which incorporation of a 1,2,3-triazole was motivated by predicted improved pharmacokinetic and clearance properties. Biodistribution and dosimetry studies in healthy nonhuman primates and humans by the laboratory of Lambin confirmed metabolic stability and relatively low uptake in the liver and gastrointestinal tract [163]. Further preclinical validation in a rat rhabdomyosarcoma model showed high tumour-to-blood ratios in which tumour uptake was shown to decrease significantly as a result of treatment with nicotinamide and carbogen and to increase significantly with 7 % oxygen breathing [160]. A feasibility study of 18F-HX4 imaging in (12) patients with head and neck cancer and (2) patients lung cancer showed a tracer uptake profile similar to that of 18F-MISO and a good correlation with CAIX immunohistochemistry, with the added advantage of shorter injection to acquisition time than with 18F-MISO [60]. Further characterization of the tracer is warranted in the evaluation of treatment response. Distinct from the nitroimidazoles, another tracer that has been studied is 64Cu-ATSM, a bisthiosemicarbazone core structure complexed with radioactive copper. The tracer is lipophilic and shows high uptake in cells, which conversely also contributes to high liver uptake which compromises its utility given that the liver is a common site of metastasis. The unfavourable distribution profile is currently being addressed by introducing structural changes to reduce lipophilicity. It is proposed that selectivity for hypoxic cells involves reduction of the Cu(II)-ATSM complex to negatively charged Cu(I)ATSM, which is trapped in cells. Recent studies have challenged the above mechanism of action and indicated that copper metabolism may also play a role in selectivity [164]. In a preclinical comparison with the nitroimidazoles, (18F-MISO, 18 F-HX4 and 18F-FAZA), 64Cu-ATSM showed the highest uptake in human head and neck carcinoma xenografts. However, it showed the lowest correlation with hypoxia-selective markers such as CAIX, casting further doubt on its specificity for hypoxia per se [165]. There are also tracers for SPECT imaging of hypoxia (reviewed elsewhere [166]) such as 99m Tc-HL91 [167, 168], but these are less widely used owing to limitations in spatial resolution associated with SPECT, rendering PET better for imaging of hypoxia. The use of these probes to broadly predict genomic instability remains to be seen. Imaging altered energy metabolism It is ironic that the imaging probe most widely used in PET, F-FDG, falls into the class of an ‘emerging’ hallmark capability – reprogrammed energy metabolism (REM). The importance of REM according to Hanahan and Weinberg [1] is 18 551 clear; the question, however, is whether it is simply another phenotype programmed by proliferation-inducing oncogenes. Cancer cells reprogram their metabolism with a shift towards increased glycolysis – a less-efficient ATP-producing pathway but with potential advantage of supplying other functions including nucleotides for cell division and generating biomass. In addition to increased expression of GLUT1 and hexokinase I/II, several oncogenes and oncometabolites are deregulated as part of the REM phenotype in glycolysis and associated pathways. These include classical activated oncogenes (e.g. PFKFB3, FASN, RAS, MYC), mutated tumour suppressors (e.g. p53 and its target TIGAR) [169], gain of function missense mutations (e.g. isocitrate dehydrogenase I/II) and germline loss of function (e.g. succinate dehydrogenase). Several of these genes and gene products interact or activate HIF1α to accentuate glycolysis under hypoxia. Subpopulations of cancer cells (hypoxic and aerobic) exist and are able to live in metabolic symbiosis through expression of different monocarboxylate transporters (MCT1 and MCT4), allowing them to make or use lactate for energy production [170]. Cells utilize the tricarboxylic acid cycle (TCA) cycle to varying extents, and the role of glutamine as a nutrient is now also well recognized [171]. Labelled glutamine, acetate and branched carboxylic acids are in development as radioprobes for investigating different aspects of REM [172]. 18F-FDG, however, remains the most sensitive tracer for imaging REM. With the advent of large-scale genomic platforms, the molecular determinants of 18F-FDG uptake are being elucidated for different tumour types. For example, the triple-negative breast cancer subtype shows particular avidity for 18F-FDG, with the ‘18F-FDG signature’ found to be associated with MYC gene copy gain, increased MYC transcript levels and elevated expression of metabolic MYC target genes [173]. A detailed overview of the clinical application of 18F-FDG is beyond the scope of this review. Table 1 provides examples of some relevant studies. The interested reader is also referred to extensive review articles on the subject [174, 175]. In addition to increased glycolytic flux, increased rates of glycogen synthesis (glycogenesis) in cancer cells have also been reported [176, 177]. A novel glucosamine analoguebased radiotracer, similar in structure to the fluorescent 2NBDG, for imaging glycogenesis has recently been reported [111]. The specificity of 18F-NFTG for glycogenesis has been demonstrated in vivo using ovarian cancer cell lines in which retention of the tracer decreases following knockdown of glycogen synthase 1 in vitro, whereas changes in 18F-FDG were unremarkable under the same conditions. Specificity of 18FNFTG has additionally been demonstrated through its increased retention upon overexpression of Rab25 that is known to increase glycogen synthesis and is correlated with glycogen levels in vitro and in vivo [111]. Unlike 18F-FDG, uptake of 18 F-NFTG is unaffected by inflammation, which would make it able to distinguish between neoplastic and inflamed tissue 552 [178]. In the ovarian cancer models used, however, the absolute uptake levels of 18F-NFTG were lower than those of 18FFDG whilst showing higher liver retention. Further characterization of this tracer in different cancer types is warranted. Adjustments in energy metabolism to fuel cell growth and division also leads to aberrant protein, lipid and phospholipid metabolism [179, 180]. Phospholipid metabolism has been probed with 11C-choline and 18F-fluorocholine. Choline uptake via CTL1 and OCT3 transporters is needed to generate phosphatidylcholine, an essential component of cell membranes. Upon entry into cells, choline kinase α catalyses its phosphorylation to phosphocholine, with eventual metabolism to phosphatidylcholine via the Kennedy pathway [181]. Uptake is increased in neoplastic tissue due to elevated choline kinase α expression [24, 182] with high expression of the enzyme associated with a worse prognosis [183]. Both choline-based tracers have found application in the clinic, primarily for the detection of locally recurrent and metastatic prostate cancer [184]. Indeed the motivation to develop choline PET in this tumour group is largely due to the failings of 18 F-FDG (low uptake, low specificity and high excretion into the bladder) which mask the signal of interest from the prostate. 11C-Choline in contrast shows limited excretion via the bladder. Both 11C-choline and 18F-fluorocholine have been shown to be better than 18F-FDG in this disease and are the most commonly used in prostate cancer imaging [185, 186]. The differences between the two choline tracers are minor: 18 F-fluorocholine appears earlier in the urinary tract. Therefore, although this excretion profile is not detrimental to the accuracy of 18F-fluorocholine, 11C-choline is preferred for imaging local recurrence [187]. Additionally, 18F-fluorocholine PET has shown utility for detection of bone metastasis, particularly in the early phase of bone metastasis [38, 188], although uptake in bone can be influenced by hormone therapy. Numerous clinical studies were published in 2014, indicating the utility of choline tracers in patient management and treatment planning in prostate cancer [26–28, 32]. Currently 11C-choline PET is approved for the diagnosis of recurrent prostate cancer in patients who have raised prostatespecific antigen (PSA) following treatment and when anatomical imaging fails to provide evidence [189, 190]. Although the majority of choline PET studies have shown significant correlation with PSA and tumour grade, the use of 11C-choline is associated with a high incidence of false-negative findings which renders it unsuitable for routine use in initial detection of prostate cancer and makes histological confirmation always necessary [189, 191]. Despite wide clinical application, 11C-choline and 18Ffluorocholine suffer from metabolic instability in vivo, being readily oxidized by choline oxidase to betaine analogues (mainly in the liver and kidneys). To overcome this problem, a deuterated version, 18F-fluoromethyl-(1,2-2H4)-choline (18F-D4-choline) was developed which shows improved Eur J Nucl Med Mol Imaging (2015) 42:537–561 resistance to oxidation whilst retaining its phosphorylation potential [181, 192]. Retention of 18F-D4-choline has been shown to be specific using three different tumour xenograft models in which uptake was associated with choline kinase α expression [193]. The radioprobe has recently been evaluated in healthy volunteers and shows a renal and hepatobiliary excretion profile and suitable dosimetry [33]. A study in NSCLC patients in the UK is underway (UK Clinical Research Network ID 13925) and findings are eagerly awaited. The development of radiolabelled acetate was motivated by interest in probing a separate arm of lipid metabolism: to chart the synthesis of fatty acids (FA) which like choline become incorporated into the cell membrane. Acetate is first transported into cells via MCTs where it becomes a substrate for acetyl-coA synthetase (ACS) which converts it to acetylcoA. Acetyl-coA is either oxidized in the mitochondria, or engages an anabolic pathway in the cytosol where it is subsequently converted to FA by FA synthetase (FASN). The latter is upregulated in cancer. Overexpression of FASN in prostate cancer has fuelled the application of 11C-acetate, both to characterize this disease and as a pharmacodynamic biomarker to verify pharmacological inhibition of FASN. Several preclinical studies have demonstrated that 11C-acetate uptake is correlated with FASN expression [194, 195]. In three prostate cancer xenografts, Yoshii et al. demonstrated that tumour acetate uptake reflected FASN expression and predicted response to orlistat, a FASN-targeted therapy [194]. In an early clinical study in patients with prostate adenocarcinoma, Oyama et al. found that 11C-acetate has better sensitivity in the detection of primary tumour than 18F-FDG (100 % vs. 83 % sensitivity. respectively) and in the detection of bone metastasis [196]. However, further studies revealed that the tracer is unable to distinguish between histologically confirmed benign and malignant lesions and thus fails to give information about cancer aggressiveness [197]. 11C-Acetate shows limited urinary excretion, which is advantageous in imaging prostate malignancies, but high uptake in the liver and myocardium. The tracer is also able to distinguish between neoplasm and inflammation better than 18F-FDG [198, 199]. Recently the laboratory of Brindle showed that imaging at later time-points is preferable with 11C-acetate to overcome the contribution of perfusion and TCA cycle intermediates to the signal whilst maximizing incorporation of the tracer into the de novo FA synthesis pathway [195]. 11CAcetate has also been used in other malignancies such as brain tumours, lung and hepatocellular carcinoma. However, its half-life of 20 min limits its wider distribution as is the case for 11C-choline. 18F-Fluoroacetate was developed to enable wider distribution though it has shown limited utility in PET oncology despite its longer half-life and rapid liver clearance [200]. Key concerns are that it is not a functional analogue of 11 C-acetate and is unstable, and has been shown in rodents to exhibit defluorination resulting in skeletal uptake [200, 201]. Eur J Nucl Med Mol Imaging (2015) 42:537–561 Though increased FA synthesis during the metabolic reprogramming in cancer has been known for some time, the role of increased FA oxidation has only recently been described [202]. A new tracer 18F-fluoropivalic acid (18F-FPIA) is thought to probe this process [203]. The radiotracer has shown high uptake in breast, prostate and brain tumour models, comparable or even superior to that of 18F-FDG [204]. Furthermore, it shows potential for brain tumour imaging with relatively low uptake in healthy brain and is currently being studied in orthotopic models of glioblastoma. It has been suggested that the tracer is likely to be a substrate for ACS and carnitine acyl transferases, but conclusive evidence for this is currently lacking and the underlying mechanisms of its trapping still require further investigation prior to clinical translation. A direct result of the reprogrammed energy requirements and glycolytic phenotype is that the tumour extracellular space becomes acidic due to elevated lactate and proton production within the cell which are pumped out as part of the cellular homeostatic controls. The extent of tumour extracellular acidosis is also determined by the level of perfusion and has implications on the other hallmarks (cell invasion, angiogenesis and metastasis) and therefore represents a useful biomarker for the characterization of malignancies and for selective drug delivery. While preclinical imaging of pH using MRI is widely reported, radionuclide imaging of pH has been rather limited [205]. A 11C-labelled probe (11C-DMO) was shown to detect pH in patients with brain tumours, based on the measurement of neutral and ionized species but is not deemed a robust method in part due to poor detection sensitivity [206]. Recently a more promising new radioprobe was reported by the laboratory of Lewis for probing tumour acidosis. Labelled with 64Cu via a NOTA chelate, the membrane-inserting pH (low) insertion peptide (pHLIP) showed preferential accumulation in tumour tissues of prostate cancer models [207]. The tracer binds as an unstructured peptide at the membrane surface at normal pH but folds and inserts across the plasma membrane in an acidic environment. The agent shows good clearance and superior tumour to tissue contrast and reduced retention in the liver, kidney and gastrointestinal tract especially when compared to an earlier version of the probe [208]. Moreover, tracer retention within the tumour correlates with other features associated with acidity such as hypoxia and lactate dehydrogenase A expression. Clinical development is eagerly awaited. Imaging of immune responses The other emerging hallmark capability – evading immune destruction – has received less attention in the radioprobe development field due to the potentially low level of exploitable biological signal it might provide. The importance of 553 immune surveillance in resisting tumour formation and progression is well recognized. Tumour growth may be enhanced, for example, by deficiencies in CD8+ cytotoxic T lymphocytes or natural killer cells, recruitment of immunosuppressive inflammatory cells such as regulatory T cells, and secretion of immunosuppressive TGF-β by tumour cells. Epidemiological evidence that ovarian [209] and colon tumours [210, 211] with high levels of cytotoxic T lymphocytes have a better prognosis support development of radioprobes for this target. In this regard, the laboratory of Radu developed 1-(2′-deoxy-2′-18F-fluoroarabinofuranosyl)cytosine (18FFAC) to monitor localized immune activation and immunosuppressive therapy by probing the deoxycytidine salvage pathway [212]. 18F-FAC is retained in proliferating CD8+ T cells relative to naive T cells due to higher expression of the solute carrier transporter SLC29a and the intracellular enzymes deoxycytidine kinase (dCK) and thymidine kinase 2 (TK2) (Fig. 1). Comparisons with 18F-FDG and 18F-FLT have shown that 18F-FAC specifically localizes in the thymus, spleen and lymph nodes, but high retention in lymphoid organs could also compromise its ability to detect weak immune responses. A further preclinical study in retrovirus-induced sarcoma using the probes 18F-FDG and 18F-FAC has shown that it is possible to differentiate between the innate and adaptive immune response because of their respective glycolytic and deoxycytidine salvage requirements [213]. A 18F-FAC analogue with improved metabolic stability and specificity for dCK, 18F-fluoro-β-L-arabinofuranosyl)cytosine (18F-LFAC), has since been developed and evaluated preclinically as a PET pharmacodynamic biomarker for dCK inhibitors [214, 215]. The findings of a 18F-L-FAC study in healthy subjects and patients with cancer and autoimmune and inflammatory diseases are yet to be reported (Clinicaltrials.gov NCT01180868). The need for tracer development to explore the complex tumour microenvironment Lastly, Hanahan and Weinberg also highlight the complexity of the tumour microenvironment. Within this environment tumour cells and various stromal cells coexist at varying abundance. These stromal cells are important in nurturing tumours and hence are important targets for radioprobe discovery. They include: CSCs, endothelial cells, pericytes, immune/ inflammatory cells including leucocytes and macrophages, CD11b+/Gr1+ myeloid progenitor cells, α-smooth muscle actin-positive cancer-associated fibroblasts and bone marrow-derived stem cells. These cell types secrete various growth factors and provide a permissive environment for tumour growth. CSCs, for example, express different receptors and no one receptor type is known to fully characterize these cell types [116]. Of the cell surface receptors, however, 554 CD133 is the most prominent and well characterized marker as discussed above and its presence is associated with the ability of CSCs to self-renew [216]. It is important to note that there are other cell surface antigens, receptors and transporters which we have not covered in detail but which may fall into one or more of the hallmarks (Fig. 1; Table 1). Examples include the overexpression of the transferrin receptor that mediates iron transport and the somatostatin receptor (SR), which mediates mitogenic signalling along with a variety of other functions. Imaging approaches have fairly recently been advanced for characterizing the transferrin receptor [217]. As a c-MYC target gene, transferrin receptor-1, and by extension, binding of its 89Zr-labelled transferrin protein radioprobe, was quantitatively associated with treatment-induced changes in MYC-regulated receptor expression in a MYC-driven prostate cancer xenograft model. It should be noted that the use of this radiotracer for estimating the transferrin receptor is context-dependent; other factors including low physiological iron concentration and HIF-1α expression also regulate receptor expression. SR which binds to somatostatin, is overexpressed particularly in neuroendocrine tumours, and SR scintigraphy has been performed widely in this tumour group using 111In-labelled octreotide (somatostatin analogue). More recently there has been a preference for PET imaging of SR with 68Ga-DOTA-TOC and 68Ga-DOTATATE, largely due to the superior imaging characteristics of PET and higher accuracy, enabling delineation of smaller lesions than with conventional scintigraphy [82, 84]. Concluding statement Tumour cells exhibit numerous properties that allow them to grow and divide. Several of these properties – both specific targets and pathway elements - are detectable by nuclear imaging methods. The high sensitivity and specificity of PET for characterizing tumours has led to its widespread exploitation for imaging several tumour phenotypes. On the one hand such methods can be used to highlight the plasticity of tumour cells in their microenvironment and permit unique tumour biology and pharmacology of novel therapies to be studied, ultimately in the species of interest (humans). However, the main drive for developing these imaging approaches remains that of managing patients. Hence the methodology will always be one that takes patient comfort and adaptation to a busy nuclear medicine/radiology clinic into consideration. This article draws upon the seminal ‘Hallmarks of Cancer’ review article by Hanahan and Weinberg in 2011 and objectively places into context the present and future roles of radiotracer imaging in characterizing tumours. We contend that there have been substantial developments in the radiotracer field including and beyond 18 F-FDG PET leading to progress in the understanding of tumour Eur J Nucl Med Mol Imaging (2015) 42:537–561 biology. New areas of nuclear imaging science include detection of mutant proteins, stem cell biology, senescence phenotype, glycogenesis and pH. These major developments are at present largely in the preclinical arena but promise to revolutionize our understanding of tumour biology when they eventually transition into the clinic. There are still areas of cancer biology, such as expression of crucial somatic mutations, where probes have yet to be developed because of low expression of the targets of interest, difficulties in specifically tracing those targets, or a lack of distinct cost effective clinical need. As the non-FDG radiotracers gain acceptance through technical and biological validation studies, it is anticipated that they will play a significant role, alongside molecular biochemical and genetic approaches in many aspects of drug development to expedite novel cancer therapeutics for patient benefit, and aid in the stratification of patients for improved outcome (patient selection) together with verification of success of such stratification methods (pharmacodynamics and response). The latter is in line with tremendous opportunities in drug development for treating patient subpopulations. While the breath of novel tracers is impressive, Table 1 indicates that, with the exception of a few studies, most imaging studies for characterizing tumours have involved very few patient groups. This is partly due to technological and regulatory challenges, as well as cost in the early stages of tracer development. 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