Prostate cancer and inflammation: A new molecular imaging challenge in the era of personalized medicine

Nuclear Medicine and Biology 68-69 (2019) 66–79

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Prostate cancer and inflammation: A new molecular imaging challenge in the era of personalized medicine☆ Orazio Schillaci a,b,1, Manuel Scimeca a,c,⁎,1, Donata Trivigno d, Agostino Chiaravalloti a,b, Simone Facchetti d, Lucia Anemona d, Rita Bonfiglio d, Giuseppe Santeusanio d, Virginia Tancredi c,f, Elena Bonanno d, Nicoletta Urbano e,2, Alessandro Mauriello d,2 a

Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Via Montpellier 1, Rome 00133, Italy IRCCS Neuromed, Pozzilli, Italy c University of San Raffaele, Via di Val Cannuta 247, 00166 Rome, Italy d Department of Experimental Medicine and Surgery, University “Tor Vergata”, Via Montpellier 1, Rome 00133, Italy e Nuclear Medicine, Policlinico “Tor Vergata”, Viale Oxford 81, 00133 Rome, Italy f Department of Systems Medicine, School of Sport and Exercise Sciences, University of Rome “Tor Vergata”, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 5 November 2018 Received in revised form 23 December 2018 Accepted 14 January 2019 Keywords: Prostate cancer Inflammation Molecular imaging Pathology Biomarkers Personalized medicine

a b s t r a c t The relationship between cancer and inflammation is one of the most important fields for both clinical and translational research. Despite numerous studies reported interesting and solid data about the prognostic value of the presence of inflammatory infiltrate in cancers, the biological role of inflammation in prostate cancer development is not yet fully clarified. The characterization of molecular pathways that connect altered inflammatory response and prostate cancer progression can provide the scientific rationale for the identification of new prognostic and predictive biomarkers. Specifically, the detection of infiltrating immune cells or related-cytokines by histology and/or by molecular imaging techniques could profoundly change the management of prostate cancer patients. In this context, the anatomic pathology and imaging diagnostic teamwork can provide a valuable support for the validation of new targets for diagnosis and therapy of prostate cancer lesions associated to the inflammatory infiltrate. The aim of this review is to summarize the current literature about the role of molecular imaging technique and anatomic pathology in the study of the mutual interaction occurring between prostate cancer and inflammation. Specifically, we reported the more recent advances in molecular imaging and histological methods for the early detection of prostate lesions associated to the inflammatory infiltrate. © 2019 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The role of inflammation in prostate cancer . . . . . . . . . . . . . . . . . 3. Molecular imaging and pathology on the way of personalized medicine . . . . . 4. Inflammation as a not redundant histological prognostic factor of prostate cancer 5. Molecular imaging of prostate cancer . . . . . . . . . . . . . . . . . . . . 6. Molecular imaging of inflammation in prostate cancer. . . . . . . . . . . . . 7. Diagnostic and therapeutic value of Immuno-PET in prostate cancer patients . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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☆ Acknowledgments: The authors wish to thank Dr. Clara Nazzaro for the technical support in data research. ⁎ Corresponding author at: Department of Biomedicine and Prevention, University of Rome “Tor Vergata”; Via Montpellier 1, Rome 00133, Italy. E-mail address: [email protected] (M. Scimeca). 1 Orazio Schillaci and Manuel Scimeca are equally first authors. 2 Nicoletta Urbano and Alessandro Mauriello are equally last authors. https://doi.org/10.1016/j.nucmedbio.2019.01.003 0969-8051/© 2019 Elsevier Inc. All rights reserved.

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1. Introduction Several molecular and genetic aspects are involved in the occurrence of prostate cancer [1]. In this context, there is growing evidence that chronic inflammation is related to the regulation of cellular events in prostate carcinogenesis, including disruption of the immune response and regulation of the tumor microenvironment [1]. Although during prostate cancer detection, both molecular imaging and histological analysis are influenced by the presence of inflammatory infiltrate, rarely diagnostic investigations take into account these conditions simultaneously. Therefore, one of the main challenges of the translational research is to identify new prognostic and predictive biomarkers for the detection of prostate lesions associated to inflammatory infiltrate. The aim of this review is to summarize the current literature about the role of molecular imaging technique and anatomic pathology in the study of the mutual interaction occurring between prostate cancer and inflammation. Specifically, we reported the more recent advances in molecular imaging and histological techniques for the detection of prostate lesions associated to the inflammatory infiltrate. 2. The role of inflammation in prostate cancer The relationship between cancer and inflammation is one of the most important fields for both clinical and translational research. Despite numerous studies reported interesting and solid data about the prognostic value of the presence of inflammatory infiltrate in cancers, the biological role of inflammation in prostate cancer development is not yet fully clarified. However, inflammation remains an integral component in prostatic diseases and may contribute to shift the balance towards tumor cell growth. The presence of inflammatory infiltrate can be observed both in benign and in malignant lesions. In benign prostate tissues, the presence of chronic inflammation classifies such lesions as prostatitis [2]. The clinical condition of prostatitis can be triggered by several causes including pathogens, physical and chemical trauma, diet, or a combination of these and other factors [3]. The main cause of chemical irritation that may cause chronic inflammation in the prostate may be represented by urine reflux or the abnormal flow of urine from the bladder back through the ureters [4]. Non-sexually transmitted pathogens such as Propionibacterium and/or E. coli are shown to cause acute and chronic prostatitis [5,6]. In addition, it is known that infections by many sexually transmitted organisms such as Neisseria gonorrhoeae [7] and Chlamydia trachomatis [8], coincides with a potential increase in prostate cancer risk. Specifically, an increased risk of prostate cancer in patients with a history of inflammatory sexually transmitted diseases provides an indirect association between chronic inflammation and prostate tumorigenesis [9]. Although the biological process of inflammatory reaction is archetypally correlated with infection, it may be triggered by autoimmune diseases, allergies, and trauma of prostate tissue. In detail, inflammatory cells can express and release several cytokines capable to activate signal cascades involved in the cell growth. Many cytokine functions and selectin-ligand interactions can be manipulated to promote growth [10]. In addition, the presence of inflammatory infiltrate contributes to increase tumor neoangiogenesis, DNA damage, cytoskeleton remodeling and extra-cellular matrix degradation to provide a substrate growth microenvironment [11]. Among the main cytokines described in prostate inflammation phenomenon, Tumor Necrosis Factor (TNF), Nuclear Factor Kappa B (NF-kB), Interleukin (IL) 1β, IL6, IL18, CCR7, CCL21 and PTX3 deserve special mention (Fig. 1). TNF is a pleiotropic cytokine involved in the stimulation of tumor angiogenesis, in the development of prostate cancer from an androgen-dependent to a castrate resistant state and, in addition, it can play a role in both epithelial to mesenchymal transition and the aberrant regulation of eicosanoid pathways [12]. TNF is mainly secreted by macrophages, T cells and natural killer (NK) cells, but non-immune cells such as fibroblasts, smooth muscle cells, and

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epithelial-cancer cells have also been reported to produce low amounts of this cytokine [13]. Emerging evidence indicates that TNF play crucial roles in both castration-induced regression of the normal prostate, as well as in prostate cancer progression to a castrate resistant state. In this context, an experimental study highlighted the role of TNF in prostate cancer displaying that after surgical castration of mice, prostates from TNF −/− mice regressed significantly more slowly than those from wild-type mice, and that regression could be restored following administration of soluble TNF [14]. More recently, Maolake et al. demonstrated that TNFα leads to the induction of CCR7 expression and that the CCL21/CCR7 axis might increase the metastatic potential of prostate cancer cells in lymph node sites [15]. These evidences could provide essential information for the use of TNFα, or cells that produce it, as early biomarkers of prostate diseases. In the process of inflammation and prostate cancer development, the role of NF??B has been appreciated. The NF??B is associated with the upregulation of tumor promoting cytokines such as IL-6 and TNF-?? [16,17]. Also, NF-κB activation affects hallmarks of cancer and inflammatory diseases through the transcription of genes involved in cell proliferation, survival, angiogenesis, inflammation, tumor promotion and metastasis [18]. From inflammatory point of view, NF-κB is a major transcription factor that regulates genes responsible for both the innate and adaptive immune response [19]. After activation, NF-κB enters the nucleus to upregulate genes involved in T-cell proliferation, maturation and development [20]. In the inflammatory response, fidelity of feedback signaling between diverse cell types and the immune system depends on the integrity of mechanisms that limit the range of genes activated by NF-κB, allowing only the expression of genes which contribute to an effective immune response and, subsequently, a complete restoration of tissue function after resolution of inflammation [21]. This is often evident in severely compromised regulation of NF-κB activity, which allows cancer cells to express abnormal cohorts of NF-κB target genes [22,23]. Activated NF-κB transcription factors were associated with numerous aspects of tumorigenesis, including promoting cancercell proliferation, preventing apoptosis, and increasing a tumor's angiogenic and metastatic potential [24]. Moreover, it is demonstrated that NF-κB can trigger the events related to mesenchymal transformation of prostate cancer cells [25]. In addition, recent studies suggest a role of NF-κB in mesenchymal transition phenomenon [26,27] and calcification production [28–30] in cooperation with osteoblast differentiation factors such as bone morphogenetic proteins and PTX3 [31–33]. All these data allow to conclude that constitutive NFκB activity within prostate cancer cells confers prognostic indications for prostate cancer tumors [34]. One of the main mediators of chronic inflammation in prostate cancer is IL6. IL6 expression mediates numerous physiological activity such as control of the acute phase response at the beginning of acute inflammation, regulation of B-cell and T-cell differentiation and activation, and support of cell growth and survival [35]. However, the initial stimuli in prostate cancer-related inflammation remain unclear. To date, it is known that blood concentration of IL-6 results elevated in patients with metastatic or castration-resistant prostate cancer and also correlates with tumor survival and response to chemotherapy negatively [36]. IL-6 is able to promote prostate cancer cell proliferation and inhibit apoptosis in vitro and in vivo. Its expression is associated with aggressive prostate cancer phenotype and may be involved in the metastatic process through regulation of epithelialmesenchymal transition and homing of cancer cells to the bone. A substantial body of evidence suggests that IL-6 plays a major role in the transition from hormone-dependent to castration-resistant prostate cancer, most notably through accessory activation of the androgen receptor. Recent evidence suggested a role in prostate cancer also for IL-1?? and IL-18, which causes a wide variety of biological effects associated with infection, inflammation, and other disease processes [37,38]. Among them, it is important to remind the presence of cytokines with antitumoral properties such as IL-10, IL-18, IL-15, and IL-21. The antitumoral effects are mainly mediated by the activation of cytotoxic T cell

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O. Schillaci et al. / Nuclear Medicine and Biology 68-69 (2019) 66–79

Fig. 1. Cytokines involved in prostate cancer development. A) Prevalence of pro-tumorigenic cytokines can sustain prostate cancer occurrence and development. B) The expression of antitumorigenic cytokines contrasts prostate cancer development. C) Table shows the manin cytokines involved in prostate tumor homeostasis.

and NK [11] (Fig. 1). The characterization of molecular pathways that connect altered inflammatory response and prostate cancer progression can provide the scientific rationale for the identification of new prognostic and predictive biomarkers. Specifically, the detection of infiltrating immune cells or related-cytokines detected by histology and/or molecular imaging techniques could profoundly change the management of prostate cancer patients. 3. Molecular imaging and pathology on the way of personalized medicine Modern medicine, based on the observation of individual clinical signs and rational conclusions, opens a new exciting era where researches, providers and patients work together to develop individualized care [39]. This innovative vision of medical sciences, called personalized medicine, has been defined by the National Institute of Health as “an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person”. It is capability to enable risk assessment, diagnosis, prevention and therapy specifically tailored to the unique characteristics of the individual, enhancing the quality of life and public health. Thus, personalized medicine has represented a decisive turning point also for prostate cancer diseases management. Molecular imaging enables early detection and/or identification of changes occurring in human body in real time, allowing researchers to explore new ways to manage and treat illnesses, thereby facilitating drug development. More than ever before, molecular imaging is playing an integral role during both initial investigations and follow-up of prostate cancer patients. In fact, molecular imaging techniques such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) use specific radiopharmaceuticals to study molecular alterations characteristics of cancer cells [39]. In particular, the spatial resolution of PET/CT offers exceptional insights into the human body that enable physicians to identify and characterize the lesions in the early stages of their development detecting, at the same time, the presence of inflammatory infiltrate [40–42]. In terms of diagnosis, molecular imaging provides the

exact location of a tumor mass and/or local recurrence, frequently before symptoms arise or abnormalities can be detected with other more invasive procedures such as biopsy or surgery. Due to the general growing inclination towards personalized medicine, the relationship between the physiochemical and biological properties of new compounds emerging from the process of drug discovery, the development itself of imaging biomarkers and quantitative imaging techniques represent a major research priority in medical communities. On the other hand, the key fora more effective diagnosis, prognosis, prediction and therapeutic management of prostate cancer could lie in the direct analysis of cancer tissue by molecular and histopathological techniques [43]. In fact, disease biomarkers have the potential to be medically valuable at all stages of the disease process from diagnosis, identification of disease subtypes and prognosis to therapeutic adjustment. In particular, analysis of prostate tissue offers the possibility to clarify the mechanisms of a prostate normal cell transformation into tumor cell and also elucidate the role of inflammation in prostate cancer progression [44]. The analysis of prostate tumor tissue obtained after surgical excision presents complex mixture of prostate cells, immune and inflammatory cells, blood vessel cells, fibroblasts, nerve cells, endothelial cells, infiltrating lymphocytes, epithelial cells, that crosstalk each other and collaborate for sustaining tumor growth and proliferation. Omic tissue analysis allows detection of tumor genetic load, tumor proteome and/or in vivo secretome alterations created by host-tumor cell interactions with inflammatory cells that may be crucial factors for tumors aggressiveness. The gradual introduction of -omic analysis on surgical specimens made the modern histopathological investigations particularly similar to molecular imaging approach. Indeed, both these disciplines are based on the study of metabolic alteration involved in cancer-inflammation crosstalk. Therefore, the creation of a collaboration between nuclear medicine and anatomic pathology department can provide an extraordinary opportunity to improve the current knowledge about the biology of the interaction occurring between prostate cancer and inflammation [45]. On note, integration of molecular imaging, histopathological and -omic data, supported by the recent innovation of artificial

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intelligence, can represent the scientific rationale on which to build new diagnostic and therapeutic protocols. The “promising alliance” between these disciplines can sustain the process of development of the modern medicine oriented towards a personalized medicine that takes into account the genetic and molecular variability of each human person. To reach this goal, a continuous and constant progress of histopathological and of molecular imaging investigations is necessary. 4. Inflammation as a not redundant histological prognostic factor of prostate cancer Prostate cancer is the most common malignancy in western men. The clinical behavior of prostate cancer is highly variable. Although prostate cancer follows an aggressive course in a significant number of men, most tumors do not cause significant clinical symptoms. Therefore, individual assessment of a tumor's aggressive potential, especially in needle biopsy specimens, helps to estimate the risk of disease progression with or without treatments with curative intent. To date, Gleason grading on prostatic biopsy is the most important predictor for biochemical recurrence, distant metastasis, and cancer-specific mortality in prostate cancer [46]. In 2005 the original Gleason grading system was initially modified [47], and more recently in 2014 [48], with the aim to improve the correlation between histological findings and treatment of the disease. Prognosis of malignancies is a crucial information, based on which optimal therapeutic decisions can be made. Inflammation may play a role in the development and progression of many cancers, including prostate cancer. Prostate cancer cases may be stratified clinically as localized disease, non-castrate rising prostate specific antigen state, non-castrate metastatic state, and finally castration-resistant state. Approximately 15% of prostate cancer patients belong to the “high risk” subset. Treatment decisions are built on the proper determination of risk level [49]. Many variables have been evaluated to identify high risk patients. Prognostic factors currently used in clinical practice of prostate cancer treatment are the Gleason score, TNM stage, prostate specific antigen level and kinetics, age, comorbidities and general condition of the patient [50]. Prediction of outcome is reliable in patients with high and low Gleason score. However, in case of intermediate scores, the prediction is less consistent, therefore other predictors, possibly more sensitive and specific during the follow-up of the clinical course and for estimating prognosis, have to be evaluated and implemented in clinical practice [51]. The value of other factors, like disease extent, defined by the percentage of positive biopsy cores was also evaluated for risk stratification [52]. However, the absence of truly reliable markers for prostate cancer diagnosis and follow-up makes it necessary to identify novel, specific, sensitive, and cost-effective biological biomarkers [53]. Local inflammatory changes are well known to be associated with the development of benign prostate hyperplasia and most probably with cancer as well, although the exact mechanisms and pathways are not fully explored and understood so far. Carcinogenesis, neoangiogenesis and metastatic capability may all be influenced by the presence of inflammation [54]. Nevertheless, the role of inflammatory process in prostate carcinogenesis remains controversial [55]. It has been hypothesized that the inflammation of prostate tissue can also be involved in the progression of cancer and if it is present in the biopsy and/or surgical specimen, may give additional clue for the determination of cancer prognosis. Publications as early as the late ‘90s found correlation between greater extent of high grade prostatic stromal inflammation in surgical specimens and postoperative biochemical recurrence-free survival, identifying inflammation as an independent predictive factor [56]. In presence of post-atrophic hyperplasia moderate to severe inflammation was found to be correlated with significant increase of prostate cancer mortality, [57]. Also, further studies confirmed that primarily chronic local inflammation in benign prostate tissue was more frequently associated with high grade than with low

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grade cancer [58]. Association of prostate cancer and inflammatory status may be responsible for poor prospect and outcome in the form of higher tumor aggressiveness and therapy resistance [59]. Inflammation may contribute to prostate cancer aggressiveness and progression by different mechanisms involving both innate and adaptive responses, different immune cells and mediators. As adaptive immune cells, for example capsular and perineural invasion, as well as biochemical progression, were related to intense infiltration of T and B lymphocytes [60,61]. It has also been found that the presence of increase CD4+ T-lymphocyte infiltrate was associated with poor prostate cancer prognosis and it can predict survival [62]. Ammirante et al. found that prostate cancer progression and metastasis is associated with inflammatory infiltration of macrophages and lymphocytes and activation of I kappaB kinase by an NF-kB independent mechanism [63]. Davidsson et al., in a case control study, showed that men with greater numbers of CD4+ regulatory lymphocytes in the environment of the prostate cancer have an increased risk of dying of prostate cancer and this identification may predict clinically relevant tumors [64]. Moreover, a systemic inflammation parameter, the neutrophil-to-lymphocyte ratio (NLR) from the white blood cells, has been considered of prognostic value in some solid tumors. However, several studies showed no association between NLR infiltrated and Gleason grading in prostate cancer metastatic patients [65,66]. Among innate inflammatory cells, also the M2 macrophages are involved in prostate cancer progression [67]. In a study, Veeranki and colleagues displayed that high density of M2 macrophages in cancer tissue is associated with higher aggressiveness and worse prognosis [60]. For what concerns molecular mediators involved in prostate cancer and inflammation, chemokines and cytokines play a role in both metastatization and progression of different tumors. Chemokines receptors are more intensely expressed on tumor cells than on normal prostate cells [60]. Chemokines can influence tumor progression by influencing the function of infiltrating inflammatory cells and acting on stromal and neoplastic cells and some of them, like CXCL8 and CCL2, have been proposed as possible prognostic markers of high grade and progressive prostate cancer [68,69]. As for the cytokines, many chemokines can play an essential role between prostate cancer risk and prostatic inflammation [70]. The hyperexpression of MIC-1 is related to cancer aggressiveness [71], higher levels of IL-6 can be registered in prostate cancer that do not express the androgen receptor and have a higher malignant potential [72]. The macrophage migration inhibitory factor (MIF) is another important cytokine involved in prostate cancer progression [73]. An increased extracellular release of MIF can bring about neuroendocrine differentiation in prostate cancer stimulating an androgen receptor independent progression [74]. Thus, the release of MIF in chronic prostatitis can favor an aggressive phenotype like the neuroendocrine one. Prostate cancer cells may also recruit mast cells and infiltrating mast cells could enhance resistance to chemotherapy and radiotherapy by activation of p38/p53/p21 and ATM protein kinase signals [75]. Presence of specific inflammatory cells expressing S100A9 such as macrophages, neutrophils and mast cells next to malignant tissue of patients with prostate cancer was associated with poor cancer-specific survival [51]. The presence of inflammatory mediators in tumor tissue and its microenvironment suggest that genetic events participating in cancer development and progression may also be found in the background of inflammatory alterations. The concept is that early genetic events generate an inflammatory process, consecutively promoting prostate cancer progression and at the same time create a stimulus resulting in a more aggressive tumor type and stage. It would be extremely useful to understand the molecular pathologic mechanisms of inflammation involved in the generation of the castration-resistant prostate cancer because treatment of chronic inflammation might provide an important therapeutic option to prevent advanced types of prostate cancer [76]. Despite these data confirm the role of inflammation in prostate cancer progression no reliable clinical prognostic marker has been identified. The role of inflammation in prostate cancer has been also evaluated in

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consideration of its influence on the response to surgery, radiotherapy, androgen deprivation therapy and in castration resistant prostate cancer patients. Data about the prognostic role of prostatic inflammation in the response to radical prostatectomy are limited in literature but some data suggest that the grade of inflammation in pre-operative biopsy specimens could be used to identify patients at risk of biochemical progression after surgery [77]. More clinical data are available in literature about the link between inflammation and response to radiotherapy in prostate cancer patients. Systemic inflammation markers have been examined and in particular NLR, platelet-to-lymphocyte ratio (PLR) and C reactive protein (CRP) which showed a significant and independent prognostic value in patients who underwent radiotherapy, either in terms of clinical progression or overall survival. [78,79]. About castration resistant prostate cancer data are consistent and both NLR and CRP resulted independent and significant predictors of overall survival and progression free survival in these categories of patients during chemotherapy [80]. Many studies consider NLR as an independent predictor of mortality in castration-resistant prostate cancer patients and support its integration in prognostic models to stratify these patients. Nevertheless, the role of systemic inflammatory changes, quantitatively characterized by the Glasgow score (deducted from C-reactive protein and albumin levels) gave promising results in estimating disease outcome, independently from other variables like age, Gleason score, comorbidity and disease stage [81]. Biomarkers related to systemic inflammatory changes, either independently or in integration with traditional parameters, are promising for the prediction of disease behavior and outcome. Many studies provide evidence that inflammation is linked to aggressive prostate cancer but further, prospective studies are needed to determine the clinical utility of inflammation as a prognostic marker of high-risk disease or as a therapeutic target. Thus, a multidisciplinary approach is essential to identify both a molecular profile and new prognostic biomarkers of prostate cancer lesions associate to inflammatory infiltrate. 5. Molecular imaging of prostate cancer Prostate cancer represents one of the most important health problems in men, with no effective treatment available in presence of an advanced state of disease. Therefore, the necessity for development of more optimal treatment modalities that could improve the patients' outcome becomes crucial. In last years, personalized medicine, where pharmaceutical therapies are individualized based on the particular characteristics of the tumor in each patient, has gained great interest [82]. In this field, molecular imaging can provide the best chance to identify prognostic and predictive biomarkers for both early diagnosis and new theranostic approaches. Currently, numerous imaging analysis are available for studying prostate cancer lesions such as magnet resonance imaging (MRI) in the form of whole-body MRI and pelvic multiparametric MRI and PET using choline as radiotracers [83]. Multi-parametric MRI emerged as a

fundamental tool for the diagnosis and correct staging of prostate cancer [84,85]. Nevertheless, developments in MR spectroscopy seem likely to permit technologies for use in clinical contexts that provide data on tumor metabolism, thus helping to establish the aggressiveness of discrete malignant foci [83]. Several data demonstrated that MP-MRI improves detection of clinically significant prostate cancer in the repeat biopsy setting or before the confirmatory biopsy in patients considering active surveillance [86]. MP-MRI is useful to guide focal treatment and to detect local recurrences after treatment. Its role in biopsy-naive patients or during the course of active surveillance remains debated. Nowadays, CT- and/or PET-based imaging modalities did not show benefit respect to MP-MRI in the imaging of early relapse of prostate. However, new radiotracers are actually under investigation and many of them are in phase I of clinical trials; the future use of these promising molecules in the diagnosis of prostate cancer could improve the utility of PET/CT analysis in the management of prostate cancer patients already in the initial phase of diagnostic path (Table 1). Indeed, PET/CT analysis, combining the molecular and morphological information of prostate lesions, represent the most appropriate molecular imagining technique to reach the goal of a theranostic protocol. A wide range of radionuclides are currently used for PET imaging analysis of prostate cancer lesions. The most commonly PET tracers used for targeting of prostate cancer are carbon-11, fluorine-18, and gallium-68, which vary in terms of their physical half-life and their chemical properties [87]. Fluorine18 is widely available, and its 110 min half-life is sufficient for production in large quantities and distribution to other sites that do not have onsite production capabilities [87]. Carbon-11 is readily produced on medical cyclotrons, but its 20 min half-life markedly limits distribution to other sites and requires significant coordination between the production and imaging team to obtain multiple patients' dosages from a single carbon-11 production [87]. Gallium-68 has a 68 min half-life and is typically produced using germanium-68 generator systems which facilitates on-site and on-demand production of [68Ga]-labeled tracers [88]. However, all PET radiotracers used for identification of biochemical recurrence showed increasing rates of positivity as the serum PSA levels rise [89–91]. Recent studies displayed an increase of the sensitivity of PSMA ligands and [18F]fluciclovine as compared to choline ligands [92]. There is little current head-to-head comparison between PSMA ligands and [18F]fluciclovine in the literature, but an early case series suggests possible superiority of [68Ga]PSMA-11 over [18F]fluciclovine for lesion detection. Despite the use of choline PET scanning is poorly accurate for the early detection of prostate cancer lesions, it is frequently used for early identification of recurrence. Thus, the most important utility of choline PET scanning seems to be in the context of rising prostrating specific antigen (PSA) following definitive local therapy. This is of particular use in patients with biochemical relapse, whereby PSA elevation is the only manifestation of their disease. Choline PET scanning can help oncologists to differentiate between localized, regional and distant prostate cancer recurrence. A study of 170 patients with biochemical failure post-radical prostatectomy showed that 44% of patients were positive at

Table 1 The main PET-radiotracers for prostate cancer. PET radiotracers

Biodistribution

Biology

[11C]Choline

Liver, spleen, pancreas, kidneys, adrenal glands, and salivary glands.

[18F]Choline [18F]Methchol

Liver, spleen, pancreas, kidneys, adrenal glands, and salivary glands. Liver, spleen, pancreas, kidneys, adrenal glands, and salivary glands, very rapid urinary excretion. Bladder, kidneys and salivary glands and duodenum.

Choline is used via a 3-step process known as the Kennedy pathway for the de novo synthesis of phosphatidylcholine, an essential component of the cell membrane; Increased uptake in prostate cancer cells. See [11C]Choline See [11C]Choline

[68Ga]PSMA

[18F]FACBC

High uptake in the pancreas and liver, lower and prolonged uptake in the Skeletal muscle and bone marrow. Low bladder excretion.

PSMA is a peptidase that is involved in the hydrolysis of N-acetyl-L-aspartyl-L-glutamate (NAAG) into the corresponding N-acetyl-L-aspartate (NAA) and L-glutamate; the expression of PSMA is 10–80 fold higher in prostate cancer respect normal tissue. Synthetic analog of the amino acid L-leucine. he structure of fluciclovine allows it to be uptaken by the tumoral cells by its amino acid transporter without incorporating in the metabolism within the body.

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[11C]choline PET analysis [93]. Summing up, choline PET analysis is commonly employed as a procedure for localizing recurrent prostate cancer in patients with biochemical relapse. It has been recently reported that in patients with biochemical relapse after prostatectomy, clinical indexes as PSA doubling time (PSAdt) and PSA velocity (PSAve) could help in the selection of those patients with low PSA levels (≤2 ng/ml) that should be subjected to [18F]Choline PET. In particular, for PSAdt ≤6 months the detection rate (DR) was 65%, and for PSAve N1 ng/ml per year the DR was 67%, thus suggesting that fast PSA kinetics could be useful in the selection of patients [94]. A disadvantage of Choline PET scan is that it only appears to be useful once a diagnosis of biochemical relapse has been made and it is currently not able to detect recurrent disease before a rise in PSA is noted (Fig. 2). In addition, literature review suggests these three known variants of choline today used, [11C]Choline, [ 18F]Fluoroethylcholine (FCH) and [ 18F]Methylcholine, are equally useful to be considered for prostate cancer imaging due to their showing similar advantages and disadvantages [95]. For this reason, the accurate knowledge of [18F]choline PET/CT bio-distribution and diagnostic pitfalls is essential [96]. Also, it is not rare to observe a consistent peritumoral inflammatory infiltrate in prostate lesions with an high uptake of [18F]FCH (Fig. 3). Correlative imaging and histological exams are often necessary to depict pitfalls. However, our experience was acquired on a large population and shows that a conspicuous amount of [ 18F]choline diagnostic pitfalls is easily recognizable and attributable to inflammation [97–99]. Despite its usefulness in the detection of recurrent prostate cancer, diagnostic imaging with [ 18F]FCH or other radiolabeled compounds is not exempt from false positive or misleading findings. In a recent study, 169/1000 (16.9%) patients showed pitfalls not owing to prostate cancer [96]. [18F]FCH uptake has been reported in other malignancies as lymphomas, colon cancers, lymphadenopathy due to bladder cancer relapse, glioma, multiple

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myeloma, colon cancer and neuro endocrine tumors. The general consensus is that an increased metabolism in these tumors is responsible of an increased uptake and metabolism of the radiolabeled compound [96]. While choline PET analysis is increasingly used for the detection of recurrent disease subsequent therapy, some studies reported interesting data about sensitivity and specificity at low PSA levels [100,101]. Consequently, there is a necessity for a consistent diagnostic examination in patients that display early biochemical failure. Currently, one PSMA ligand was approved by the Food and Drug Administration (FDA), the radiolabeled anti-PSMA antibody capromab pendetide (ProstaScint), which has a low accuracy for prostate cancer detection, as it is a large antibody that binds to the intracellular domain of PSMA [102]. On the other hand, there are a few small molecules PSMA ligands that bind to the active site in the extracellular domain of PSMA, thus providing increased tumor uptake and high image quality [103]. These small-molecule PSMA ligands bind to the active site in the extracellular domain of PSMA and are internalized and endosomally recycled, leading to enhanced tumor uptake and retention and high image quality. The two first PSMA agents for PET imaging were [ 18F]DCFBC and [ 68Ga] PSMA-11, followed by another two probes with theranostic capabilities, the chelator-based PSMA-617 and the PSMA inhibitor for imaging and therapy PSMA-I&T [104]. Later, some second-generation [ 18F]labeled PSMA ligands were introduced to overcome the high blood-pool activity and low tumor-to-background ratios of [ 18F]DCFBC, namely, [ 18F] DCFPyL and, more recently, [ 18F]PSMA-1007, which has very low urine clearance. The most widely used [ 68Ga]labeled PSMA ligands for PET imaging are [ 68Ga]PSMA-11 ([ 68Ga]PSMA-HBED-CC) and the theranostic agents [ 68Ga]PSMA-617 and [ 68Ga]PSMA-I&T [105]. [ 18F] labeled agents include [ 18F]DCFBC [106], [ 18F]DCFPyL [107], and [ 18F] PSMA 1007 and [ 18F]FACBC [108]. [ 18F]FACBC (fluciclovine or

Fig. 2. [18F]choline uptake and immunohistochemical analysis. A,B) Focal uptake of [18F]choline in an osteoaddensant lesion of the left thighbone in a patient with recurrence of prostate cancer after radical prostatectomy (PSA 0.98 ng/ml, PSA doubling time 1.3 months). C,D,E) Representative images of bone metastatic lesions showing PSA positive prostate cancer cells.

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Fig. 3. [18F]Fluoroethylcholine PET and immunohistochemical analysis of a prostate cancer patients. A) [18F]Fluoroethylcholine PET scan shows high uptake in the prostate; SUV max 31.18 – SUV average 25.39. B–G) Immunophenotypical characteristics of the prostate cancer. B) Immunohistochemical analysis displays no/rare PDL1 expression by prostate cancer cells. C) Image shows high peritumoral CD3 infiltrate. D) Several CD4 positive lymphocytes in the peritumoral site. E) Numerous CD8 positive lymphocytes in the peritumoral site. F) CD68 positive tumor-associated macrophages. G) CD163 positive tumor-associated macrophages.

Axumin™) is a radiolabeled amino acid analog that is accumulated after preferential uptake by prostate tumor cells, because it does not undergo further metabolism in the cells [109]. Fluciclovine PET was found to be successful in the assessment of primary and metastatic prostate cancers [110,111]. Fluciclovine PET effectively localized the source of increased PSA in patients with biochemical recurrence. In a meta-analysis evaluating 6 articles and 251 patients with biochemical recurrence, the pooled sensitivity and specificity of fluciclovine PET on a per-patient analysis were 87% and 66%, respectively [112]. These prostate cancer-targeted PET tracers are not specifically targeting to prostate tumor cells, but are worthy of attention in this review, because they are promising competitors as prostate cancer-targeted PET tracers. [ 68Ga]PSMA-targeted diagnostic imaging has been recently developed [113]. Therefore, the targeting of PSMA has become increasingly important over the last decade. PSMA-PET modality has many advantages over traditional PSA testing, specifically the capability to identify the exact location of lymph node metastases and distant soft tissue spread. Salvage radiation therapy has been delivered to the prostatic site without knowledge of the localization of residual disease. This new modality opens the possibility of salvage surgical or radiation therapy that can be targeted to the correct location. In a very recent study, Komek and colleagues provided evidence about the potential utility of tracer uptake (SUV) cut-off values on [ 68Ga]PSMA PET/CT in the identification of the survival outcome of patients with metastatic disease and thereby in assisting the selection of individualized therapeutic strategies tailored to the expected prognosis [114]. In our experience, the uptake of [68Ga]PSMA in a primary lesions and metastatic sites is a good predictor of histological aspects of prostate cancer (Fig. 4). Despite the significant progress in molecular imaging techniques, early detection of prostate cancer can be made difficult for the presence of cancer associated

inflammatory infiltrate. An accurate and specific in vivo detection of inflammatory infiltrate by molecular imaging analysis could ameliorate our knowledge about the relationship between cancer and inflammation laying the foundation for new diagnostic and therapeutic strategies. 6. Molecular imaging of inflammation in prostate cancer Inflammation plays a significant role in many disease processes. Recently, technological advance in molecular imaging offers new insight into the diagnosis and treatment evaluation of various inflammatory diseases and diseases involving inflammatory process such as prostate cancers [115]. In particular, it is known that non-physiological inflammatory reactions or delay in the resolution of inflammation can induce damage at normal cells in the tissue triggering molecular events related to carcinogenesis. [ 18F]FDG (2-deoxy-2- 18F-fluoro-D-glucose) is certainly the most widely used PET imaging tracer and has been applied successfully in tumor detection, staging, and therapy evaluation, as well as in cardiovascular and neurological diseases [116]. In inflammatory diseases, [ 18F]FDG PET also has its value. Nevertheless, [ 18F]FDG PET imaging of inflammation tends to give false-positive results, especially in patients with cancer. Consequently, new imaging tracers and targets for more specific inflammation detection and therapy evaluation are under intensive investigation. PET imaging with these new tracers greatly improved our understanding of the mechanisms in which inflammation participate to cancer occurrence and development. Several investigations displayed that TNF-α is important in acute and chronic inflammatory disorders associated to prostate cancer [15,117]. Previously, Cao et al. tested a PET tracer [ 64Cu]DOTA-etanercept, to image acute inflammatory process induced by tetradecanoyl phorbol

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Fig. 4. [68Ga]PSMA uptake and histological analysis in a prostate cancer patient. A) Maximum Intensity prohjection (MIP) image showing [68Ga]PSMA uptake in multiple lymph nodes, lung nodules and in the sternum of a patient affected by prostate cancer after radical prostatectomy (PSA 12 ng/ml). B) Hematoxylin and eosin section of prostatectomy. C) 35ßH11 expression in prostate cancer cells. D) PSA positive prostate cancer cells.

acetate [118]. Micro-PET imaging displayed high [64Cu]DOTA-etanercept uptake in the inflamed ear only during the early acute inflammatory phase but not the chronic inflammation phase, indicating that TNF-α contributes to the onset of acute inflammation. Therefore, [ 64Cu]DOTAetanercept could have a role in the early detection of acute inflammatory injury that may precede the onset of prostate cancer. In addition, in vivo detection of TNF-α-related prostate inflammation opens the way for new theranostic opportunities. In fact, therapies based on the use of anti-TNFα antibodies represent a concrete reality for patients affected by rheumatoid arthritis (RA) [119] or inflammatory bowel disease [120]. NF-kB upregulation in prostate cancer is accompanied by the enhanced recruitment of inflammatory cells and production of proinflammatory and pro-cancerogenic cytokines, such as IL-1, IL-6, IL-8, and TNF at sites of inflammation and malignant transformation. Experimental study of Vykhovanets and colleagues reported interesting evidence about the possibility to use NF-kB molecular imaging as a target of prostate inflammation [121]. By using fluorescent NF-kB molecules authors performed in vivo imaging analysis that allowed them to elucidate mechanisms involved in the association between NF-kB expression and prostate cancer. The molecular imaging analysis of NF-kB activity could be an innovative method to characterize the role of cytokineinduced NF-kB signaling in prostatic inflammation and prostate cancer development. Also, due to the existence of several NF-kB inhibitor, such as dexamethasone, NF-kB in vivo imaging might be a valuable tool to screen possible candidate drugs for treatment of tumor associated inflammation. Although not yet applied to the prostate, very recent investigation highlighted the possibility to use molecular imaging analysis capable to detect ILs expression or their receptors. In a paper published in 2018, authors developed a new methods to investigate the expression of IL2R by using [ 18F]FB-IL2v [122]. Similar methods could be used to study the role of IL1, IL6 and IL8 in prostate cancer.

The application of molecular imaging analysis, especially PET/SPECT investigation, for the study of relationship between inflammation and prostate cancer will be able to profoundly change our knowledge about cancer occurrence and progression. One of the most important applications of imaging inflammation in cancer is the targeting of the upregulation and trafficking of immune system cells as they interact with normal or cancer prostate cells. For many years, targeting of immune cells was mostly isotope-based, with SPECT and PET applications. More recently, MRI-based approaches have gained momentum. Among the new targets currently used for the targeting of immune system cells, Somatostatin receptor (SSTR) has been investigated [123]. SPECT imaging of SSTR expression in neuroendocrine tumors has been wellestablished for lesion detection and therapeutic monitoring. In addition, high levels of SSTR expression was found on activated lymphocytes and macrophages [123]. Various synthetic somatostatin agonists able to bind to the receptors have been synthesized during the past two decades for diagnostic and therapeutic purposes. The presence of a specific class of receptor on cell's surfaces should give a potentially biological target to be used for therapy [123]. False positivity to SSTRs expression was considered when localizations with a suspicious uptake not were confirmed by other radiologic procedures. In particular, inter-mass uptake could be related to the presence of infiltrated tumor cells. Translocator protein (TSPO), typically located in the outer mitochondrial membrane, is mainly responsible for transporting cholesterol across the membrane for cell signaling and steroid biosynthesis [124,125]. Previous studies found high TSPO expression in macrophages, neutrophils, lymphocytes, activated microglia, and astrocytes [126–128]. Although mainly investigated as biomarkers of brain inflammation, TSPO-SPECT and PET analysis could be a powerful tool for the study of tumor associated inflammatory infiltrated. Tantawy et al. recently provide the evidence that the TSPO radio ligand, [ 18F]VUIIS1008, can be used for the early identification of primary prostate lesions. Indeed, in vivo

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experiments demonstrated that TSPO expression in the tumors of Pten/ Trp53 mice was regionally heterogeneous, as confirmed by immunohistochemistry, but it was consistently observed across tumors, as determined using [18F]VUIIS1008 [125]. Macrophages are today considered the major drivers of inflammatory insult. For many solid tumors, such as the cancer of the prostate, breast and lung, high density of cells expressing macrophage-associated markers have generally been found in association with a poor clinical outcome, characterized by inflamed microenvironment, a high level of dissemination and resistance to conventional chemotherapies [129]. Tumor associated macrophages (TAMs) support prostate cancer cell proliferation most commonly via NF-κB, which is correlated with resistance to apoptosis, angiogenesis, and metastasis [130,131]. M2 polarization is fundamental for the creation of a pro-tumor microenvironment. Among involved factors, overexpressed cathelicidin-related antimicrobial peptide-mediated differentiation and polarization of early myeloid progenitors into protumorigenic M2 macrophages during prostate cancer progression [132]. At least two classes of iron oxide nanoparticles for MRI are currently under evaluation in preclinical testing. Superparamagnetic iron oxide nanoparticles (SPIO) have a hydrodynamic diameter of 60–180 nm, while ultrasmall SPIO (USPIO) have a diameter of 20–50 nm, both of which have been extensively evaluated in vivo [133]. Intravenously injected SPIOs are quickly phagocytosed by macrophages, and are commonly too large to cross the endothelium of tumor microvessels in significant quantities to allow detection with MR imaging. In cancer tissues infiltrated by TAMs, nanoparticles are slowly phagocytosed by macrophages, and nanoparticles retained in TAMs in turn exert a T2-signal effect on delayed MR scans, several hours to days after nanoparticle administration [134]. Intracellular iron oxides exert only minimal or no T1 effect due to lack of interactions with protons [135]. This “decoupling” of T1- and T2-signal effects can be used as a noninvasive imaging indicator for TAM phagocytosis of the USPIO. Once within cells, nanoparticles undergo a slow metabolization; thus, baseline MR signal intensities of tumors and macrophage infiltrates are regained after several days or weeks. For what concern the identification of TAMs by PET-SPECT/CT analysis several radiotracers have been developed. One of the most promising radiotracer for the identification of macrophages has been described in a paper of Pérez-Medina and colleagues. In particular, authors developed 89Zr-labeled TAM imaging agents based on the natural nanoparticle reconstituted high-density lipoprotein (rHDL) [136]. In an orthotopic mouse model of breast cancer, authors showed the specificity of [89Zr]rHDL for macrophages. Histologic and immunohistochemical analysis showed a significant colocalization of radioactivity with TAM-rich areas in tumor sections. Quantitative macrophage PET imaging with [ 89Zr]rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment [136]. Similar to TAMs, tumor infiltrated neutrophils and lymphocytes have an important role in both prostate cancer occurrence and progression. NLR is a widely used, representative marker of systemic inflammatory response within the body. High NLR is a negative prognostic factor in a variety of malignancies including urological tumors and in particular prostate cancers [137]. Indeed, preoperative NLR ≥ 3 was associated with aggressive prostate cancer. In line with this, preoperative NLR may still be useful in selected patients to identify aggressive prostate cancer helping patient selection for active surveillance protocols. Thus, imaging techniques that allow in vivo evaluation of NLR could improve the management of prostate cancer patients. In recent years many radiolabeled molecules are been proposed for the monitoring of tumor infiltrating lymphocytes. At present, numerous T cell tracking approaches have been developed by using noninvasive molecular imaging technologies that consent the researchers to detect the delicate biological/biochemical processes of the adoptive T cells in human subjects in vivo. The main aim of these techniques is to early noninvasively track of the infused tumor-specific T cells,

and to study the biodistribution, mechanism and function of tumorspecific T cells for defining the efficacy of the T cell therapy in a timely manner and assisting decision-making in clinical trials [138]. Despite rapid progression in this field, we still face challenges in developing safe and reliable methods for noninvasive tracking of the infused T cells in patients. Thatindium-111 [ 111In]oxiquinolon and technetium- 99mhexamethylpropylene amine oxime (99mTc-HMPAO) have been a clinical routine for ex vivo labeling of autologous leukocytes for detecting infections and inflammations [139]; yet until now few radiopharmaceutical tracking methods surpass them in clinical settings [139]. In optical fluorescence imaging, T cells are labeled by fluorophores, fluorescent proteins, or quantum dots (QDs). The fluorophores are usually near infrared (NIR) fluorescent dyes, such as indocyanine green, Cy5.5, IRDye800CW, VT680, and the Alexa Dye Series [138]. MRI is widely used in clinical practice. MRI cover a wide range of medical practice, including diagnosis, functional and anatomical investigations of progression of diseases. Some characteristics make MRI an ideal method to other modalities in many cases. For example, the imaging process does not involve ionizing radiation; and it yields the best soft tissue contrast among all imaging modalities. Four classes of MR contrast agents have been developed: a) Positive contrast agents containing paramagnetic gadolinium (Gd) complexes, b) Negative contrast agents containing superparamagnetic iron oxide (SPIO) nanoparticles, c) Chemical exchange saturation transfer (CEST) probes, and d) [18F]containing probes [138]. PET/SPECT has been the only accepted tool for tracking the T cells in both preclinical animal models and human trials. Direct radiolabeling of T cells is relatively simple and straightforward. In direct ex vivo labeling, T cells can be label with [18F]FDG before injection to give the PET signals and study their distribution. This method displays the biodistribution and trafficking of [ 18F]FDG labeled T cells allow a quantitative evaluation of T-Cells in the body and in particular in the tumor mass [138]. Another opportunity to track cancer lymphocytes is offered by the labeled of these with [ 64Cu]pyruvaldehyde-bis(N4-methlthiosemicarbazone) ([64Cu]PTSM). Once in the cell, the reduction of Cu(II)-PTSM complex gives rise to a dissociated Cu(I) ion, which is trapped in the cell due to the Cu(I) ion charge [140]. The result showed [ 64Cu]PTSM had higher labeling efficiency than [18F]FDG, but had a similar efflux rate. As concern the possibility to label T-Cell in vivo several PET tracers are available. A significant progress was made towards developing novel probes specifically targeting the activated T cells. Among these, a nucleoside analog, 1(2′-deoxy-2′-[ 18F]fluoroarabinofuranosyl) cytosine ([ 18F]FAC), have been screened and identified. It had enhanced retention in proliferating tumor T cells [138]. The clinical trials have been completed for determining the biodistribution of [ 18F]FAC in patients with blood cancers, solid tumors and autoimmune diseases [141]. Although all molecules here cited have the potentiality for the use in the management of prostate cancer patients, no clinical application have been approved. Therefore, the identification and development of specific radiolabeled molecules for the recognition of inflammatory cytokines or inflammatory cells could implement the possibility for diagnosis or cure of prostate cancer patients. 7. Diagnostic and therapeutic value of Immuno-PET in prostate cancer patients Immuno-PET is an emerging technique that combines the specificity of monoclonal antibodies with the high sensitivity and quantitative potential of PET to non-invasively identify disease, stage, and response to therapy [142]. Immuno-PET targeting of cytokines and/or lymphocytes can provide spatial and temporal information that is currently unavailable using the standard techniques [143]. Antibodies with high affinity and specificity can be conjugated to radionuclides, and PET imaging can be used to non-invasively monitor and quantify monoclonal antibodies distribution in real time [144]. Intact antibodies function well as therapeutics due to their long serum half-life (1–3 weeks), increasing

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exposure of the affected tissues to the antibody; furthermore, biological activity from the effector domain (fragment crystallizing; Fc) is often essential for therapeutic function [145]. In contrast, the long half-life of intact antibodies hampers their use as imaging agents: several days are required for blood and background clearance in order to achieve a good signal/noise ratio. For radiolabeled antibodies, this means that the organism will be exposed to radioactivity for an extended period of time. Furthermore, biological activity is undesirable in an imaging agent, in the interest of studying the system without perturbing it and reducing the risk of unwanted side effects [146]. Many of these issues have been addressed by enzymatic cleavage or reformatting antibodies into smaller antibody fragments with a variety of molecular weights, valencies, clearance routes, and conjugation strategies. Using singlechain variable fragments (scFv; 25 kDa) and portions of the constant region (CH) as building blocks, additional fragments can be constructed such as diabodies (Db; dimers of scFv, 50 kDa), minibodies (Mb; dimers of scFv-CH3, 80 kDa), and scFv-Fc (dimers of scFv fused to Fc, 105 kDa) [147,148]. Comparisons of the pharmacokinetics and targeting of intact and antibody fragments (enzymatically-derived and recombinant) have been conducted; recent examples include targeted imaging of prostate-specific membrane antigen (PSMA) using [111In]radiolabeled F(ab')2 and Fab (Fragment antigen binding) fragments, and [ 89Zr] labeled minibodies and diabodies with small animal SPECT or PET [149]. Continued interest in smaller antibody fragments and protein scaffolds is evidenced by several imaging studies with nanobodies [150] and affibodies [151]. Antibody-based imaging agents have several advantages including their naturally high avidity, antigen specificity, and ease of production. Thus, immuno-PET imaging can provide a means to noninvasively assess antibody drug pharmacokinetics and the expression of cell surface markers of disease [152]. In the management of prostate cancer patients, immuno-PET provides an exciting new technology but its application is still limited due to the presence of few molecular target validated in large cohort studies. Among the markers emerged in the field of prostate cancer immune-PET there are programmed deathligand 1 (PD-L1) or PD-1 expression [153,154] and TNFα, and PSMA molecules [155]. Although PD-L1 expression has been linked to poor prognoses and better therapeutic responses, its true predictive value is still unknown [156]. PD-L1 expression in prostate cancer validation has been confounded by ex vivo results from histologic studies using

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different antibodies and positive or negative staining thresholds [157]. In our laboratory, we frequently observed PD-L1 prostate cancer lesions in patients with high [ 18F]FCH uptake and multiple metastatic foci (Fig. 5). The therapeutic opportunities offered by the use of specific anti-PD-L1 antibodies open the way for the development of molecular imaging protocol. In fact, there is an explicit need for molecular imaging tools that can noninvasively capture and quantify the spatiotemporal PD-L1 prostate expression profiles. For these reasons, early attempts at immune checkpoint imaging have used antibody scaffolds including [ 111In] radiotracers directed against murine and human PD-L1 for SPECT imaging of prostate, breast and non–small cell lung cancer [158–160]. Heskamp et al. were one of the first to provide evidence that non-invasive imaging of PD-L1 in the tumor with a mAb-tracer is technically possible [161]. Authors used an [ 111In]labeled IgG1 mAb (PD-L1.3.1), which specifically and exclusively binds human PD-L1 with a nanomolar affinity. Using an [ 89Zr]labeled anti-mouse-PD-L1 mAb, upregulation of PD-L1, after radiotherapy alone or in combination with anti-PD-1 therapy, was monitored in head-and-neck squamous cell carcinoma and melanoma mouse models [162]. PET/CT correlated to flow cytometry showed upregulation of PD-L1 in the irradiated tumors but not in the anti-PD-1-treated tumors [162]. In addition, it is known that there is a correlation between the response to PD-L1 therapy and the presence of PD-1 positive tumor-infiltrating lymphocytes [163,164]. Thus, imaging of PD-1 has also received attention, albeit to a lesser extent than imaging of PD-L1. Natarajan et al. evaluated a mouse anti-PD-1 mAb (eBioscience) radiolabeled with [ 64Cu] through DOTA, showing efficient binding to PD-1positive cells both in vitro and in vivo [165]. Recently, England et al. evaluated the FDA-approved mAb pembrolizumab, a humanized IgG4 mAb, using [ 89Zr]labeling for PET imaging [166]. Imaging in mice implanted with human peripheral blood mononuclear cells confirmed its specific binding to human PD-1 positive T-cells in salivary glands. On the other hand, Cole et al. were the first to evaluate [ 89Zr]labeled FDA-approved anti-PD-1 mAb nivolumab for PET imaging in non-human primates. Nivolumab is a fully human IgG4 that binds with similarly high affinity (3 nM) to both human and cynomolgus PD-1. CTLA4 counteracts the activity of the T cell co-stimulatory receptor CD28 and actively delivers inhibitory signals to the T cell, with the final downregulation of T cell activation through several mechanisms (e.g., increasing the T cell activation threshold and attenuation of clonal expansion) [167]. In addition to its

Fig. 5. PET and immunohistochemical analysis of a prostate cancer patient with multiple metastatic nodules. A,B) [18F]Fluoroethylcholine PET scan shows high uptake in both the prostate and several bone sites. C) Image shows high uptake in the prostate; SUV max 39.15 – SUV average 28.49. D) Head scan displays a small brain metastatic lesion. E) Immunohistochemical analysis displays high PDL1 expression in prostate cancer cells. F) No/rare CD3 positive lymphocytes in the prostate cancer lesion. G) No/rare CD68 positive macrophages in the prostate cancer lesion.

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expression on T cells, CTLA4 may also be expressed by many tumor types, such as non-small-cell lung cancer, although the biological consequences remain to be elucidated. CTLA4-targeted antibodies have shown efficacy in the treatment of many cancers, and many of these antibodies have been approved by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical use [168]. Targeting CTLA-4 with a monoclonal antibody (mAb) results in transient enhancement of T cell responses and tumor eradication in preclinical studies [169]. Tremelimumab and ipilimumab are humanized mAbs that target CTLA-4 (anti-CTLA-4). Ipilimumab was the first immune checkpoint agent to be granted FDA-approval after demonstrating an improvement in the overall survival of patients with metastatic melanoma [170]. In preclinical prostate cancer models, anti-CTLA-4 monotherapy has been largely unsuccessful, but it has been shown to be efficacious when combined with other treatment modalities, including surgery [171], cryoablation [172], and vaccines [173], as these therapies may enhance tumor antigen release that potentiates the therapeutic effects of anti-CTLA-4. Most clinical trials of ipilimumab in prostate cancer have been conducted in the advanced castration-resistant setting. In this context, recently [ 64Cu]DOTAipilimumab has been developed and test on in vivo model of lung cancer [174]. Numerous evidences demonstrated the role of the cytokine TNFα in both prostate cancer occurrence and progression [13,14]. Thus, the early identification of TNFα expression by molecular imaging analysis by using 99mTc-anti-TNF-α scintigraphy could provide essential information for the management of prostate cancer patients. At the state of art, 99mTc-anti-TNF-α scintigraphy is already applied to study inflammation state of joints in RA patients [175]. However, it is important to remember that actually, the main application of Immuno-PET in prostate cancer concern the use of anti-PSMA antibodies. PSMA is a non-soluble type 2 integral membrane protein with carboxypeptidase activity, expressed on the apical surface of endothelial cells [176]. This molecule is express in the cytoplasm of normal prostate cells but it migrates on the membrane during malignant transformation. Membrane over-expression of PSMA is associated with higher prostate cancer grade and androgen deprivation, further increasing in metastatic disease and when castration resistance sets in [177]. These evidences suggest that PSMA plays an essential role in prostate cancer progression, but the correlation with Gleason Score and serum value of PSA is not well established as yet [178,179]. The role of Immuno-PSMA-PET for primary staging of prostate cancer is less well defined as the potential important role for biochemical recurrence after treatment with curative intent. The detection rate of intraprostatic PSMA-positive lesions should be up to 95% (when primary lesions are separately analyzed as discussed above). Nevertheless, sensitivity for detection of every intraprostatic cancer focus (i.e. pathological confirmed tumor localization) remains relatively low with a pooled sensitivity around 70% and a specificity around 84% [180]. J591 is a humanized monoclonal antibody (mAb) that targets the extracellular domain of PSMA [181,182]. After binding PSMA, the J591-PSMA complex is rapidly internalized into prostate cancer cells [183]. The labeling of J591with radio molecules as zirconium made this antibody suitable for in vivo molecular imaging analysis. Recently, Pandit-Taskar et al. [184] evaluated the safety, biodistribution, and kinetics of [ 89Zr]huJ591 in patients with metastatic prostate cancer. The positive results of this study candidate the [89Zr]huJ591 as a promising radiolabeled molecule for the management of prostate cancer patients affected by bone metastasis. Today, immuno-PET represents a very promising tool for personalized medicine in the context of multimodality treatment strategies. Solid preclinical and clinical studies have been performed showing the safety, the improved image quality, as well as the potential for proper estimation of the antigenic expression level of immuno-PET laying the foundation for its use in the management of prostate cancer patients. Furthermore, it is attractive for studying the in vivo behavior of antibody-based therapies and for better understanding their therapy efficacy.

8. Conclusions The presence of inflammatory infiltrate in prostate cancer is frequently associated with poor prognosis and resistance to therapy. In line with these considerations, the study of the mutual interaction between cancer cells and immune system in prostate cancer represents one of the hot topics of scientific community. Therefore, the identification of molecular mechanisms involved in cancer-inflammation “crosstalk” will allow to find biological targets for both ex vivo (biopsy) and in vivo detection of prostate lesions. In this context, the Anatomic Pathology and Imaging Diagnostic teamwork can provide a valuable support for the validation of new targets for diagnosis and therapy of prostate cancer lesions associated to inflammatory infiltrate. Even if this strategy could appear to be expensive in terms of health care costs in the short term, in our opinion the high costs of nuclear medicine procedures as compared with histological analyses, can be amortized in the long run. Specifically, the benefits in the management of prostate cancer patients, mainly in the choice of more appropriate anti-tumoral therapy, will have a positive impact on direct health costs and primarily will have a significant impact on the patients' quality of life. Funding No funding was received. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. References [1] Inamura K. Prostatic cancers: understanding their molecular pathology and the 2016 WHO classification. Oncotarget 2018 Feb 16;9(18):14723–37. [2] Khan FU, Ihsan AU, Khan HU, Jana R, Wazir J, Khongorzul P, et al. Comprehensive overview of prostatitis. Biomed Pharmacother 2017 Oct;94:1064–76. [3] Stark T, Livas L, Kyprianou N. Inflammation in prostate cancer progression and therapeutic targeting. Transl Androl Urol 2015 Aug;4(4):455–63. [4] Kirby RS, Lowe D, Bultitude MI, Shuttleworth KE. Intra-prostatic urinary reflux: an aetiological factor in abacterial prostatitis. Br J Urol 1982;54:729–31. [5] Shinohara DB, Vaghasia AM, Yu SH, Mak TN, Brüggemann H, Nelson WG, et al. A mouse model of chronic prostatic inflammation using a human prostate cancerderived isolate of Propionibacterium acnes. Prostate 2013;73:1007–15. [6] Elkahwaji JE1, Zhong W, Hopkins WJ, Bushman W. Chronic bacterial infection and inflammation incite reactive hyperplasia in a mouse model of chronic prostatitis. Prostate 2007;67:14–21. [7] Pelouze PS, editor. Gonorrhea in the Male and Female: A Book for Practitioners. Philadelphia: W. B. Saunders Company; 1935. [8] Poletti F, Medici MC, Alinovi A, Menozzi MG, Sacchini P, Stagni G, et al. Isolation of Chlamydia trachomatis from the prostatic cells in patients affected by nonacute abacterial prostatitis. J Urol 1985;134:691–3. [9] Hayes RB, Pottern LM, Strickler H, Rabkin C, Pope V, Swanson GM, et al. Sexual behaviour, STDs and risks for prostate cancer. Br J Cancer 2000;82:718–25. [10] Koong AC, Denko NC, Hudson KM, Schindler C, Swiersz L, Koch C, et al. Candidate genes for the hypoxic tumor phenotype. Cancer Res 2000;60:883–7. [11] Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7. [12] Tse BW, Scott KF, Russell PJ. Paradoxical roles of tumour necrosis factor-alpha in prostate cancer biology. Prostate Cancer 2012;2012:128965. [13] van Horssen R, Ten Hagen TL, Eggermont AM. TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility. Oncologist 2006 Apr;11 (4):397–408. [14] Davis JS, Nastiuk KL, Krolewski JJ. TNF is necessary for castration-induced prostate regression, whereas TRAIL and FasL are dispensable. Mol Endocrinol 2011 Apr;25 (4):611–20. [15] Maolake A, Izumi K, Natsagdorj A, Iwamoto H, Kadomoto S, Makino T, et al. Tumor necrosis factor-α induces prostate cancer cell migration in lymphatic metastasis through CCR7 upregulation. Cancer Sci 2018 May;109(5):1524–31. [16] Nguyen DP, Li J, Yadav SS, Tewari AK. Recent insights into NF-κB signalling pathways and the link between inflammation and prostate cancer. BJU Int 2014 Aug;114(2):168–76.

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