A β probe for radioguided surgery: from the design to the first preclinical tests

Transcription

1 A β probe for radioguided surgery: from the design to the first preclinical tests Scuola di Dottorato in Scienze Astronomiche, Chimiche, Fisiche e Matematiche Vito Volterra Dottorato di Ricerca in Fisica XXVIII Ciclo Candidate Andrea Russomando ID number Thesis Advisor Prof. Riccardo Faccini Co-Advisors Dr. Silvio Morganti Dr. Elena Solfaroli Camillocci A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics October 12 th, 2015

2 Thesis not yet defended Andrea Russomando. A β probe for radioguided surgery: from the design to the first preclinical tests. Ph.D. thesis. Sapienza University of Rome andrea.russomando@roma1.infn.it

3 Qui si fa scienza, mica fantascienza S. Morganti

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5 Contents 1 The treatment of cancer Cancer basics Diagnostics Computed tomography Magnetic resonance imaging Nuclear Imaging (PET and SPECT) Intraoperative localization of cancer cells Surgical navigation Fluorescence-guided surgery Radioguided surgery Use of β+ decay Use of β- decay Medical applications Radiotracers Radioactive isotopes Carriers Radiotracer for β -RGS Clinical cases of interest Brain tumours Uptake evaluation: meningiomas and gliomas Neuroendocrine tumours Uptake evaluation: NETs Scintillator s characterization Doped para-terphenyl Stability to temperature variation Light attenuation length Light collection Crystal height optimization Sensitivity to photons Probe design Guidelines Probe First test with liquid Yttrium Probe v

6 4.3.1 After-pulse Field of view Second test with liquid Yttrium ProbeSiPM Minisipm Evaluation of detector performances Development of the phantoms Validation of the phantoms Realization of real topologies with phantoms Minimum detectable tumour residual Spots identification Human feedback Simulation Monte Carlo False positive and false negative probability Performance on clinical cases Ex-vivo specimens Development of a multichannel probe Design of the imaging probe Pixelated crystal Single crystal Isolated spot identification Spots pair identification Conclusions 103 Bibliography 105 vi

7 Introduction In this Thesis, a novel approach to Radio Guided Surgery is discussed. Radioguided surgery is an established technique within the field of oncology surgery. In this technique a γ radio-marked tracer, a substance that is preferentially uptaken by tumour cells, is administered to the patient before the surgical operation. A nuclear probe provides the surgeon a precise information about the distribution of a radioactive labelled structure improving the surgery outcome, minimizing the surgical invasiveness thus maximizing benefit to the patient. In this Thesis, an innovation of the radio-guided surgery exploiting β emitters is investigated. The characteristics of this radiation allow the possibility to extend the technique even to cases with a large uptake from surrounding healthy organs. This new procedure is expected to bring big benefits especially when the diseases hit critical organs, like the brain. In this cases sometimes an intervention could be not considered due to the worst cost benefit ratio. In cap. 1 general informations about cancer and diagnostic techniques are given. Ways to assist the surgeon in a complete tumour resection are also investigated, focusing on RGS. In cap. 2 a description of the selected radiotracer, DOTATOC marked with 90 Y, and the cases analysed for the first application of the β -RGS are reported. In cap. 3 detailed studies about the scintillator selected to develop the beta probe are proposed. In cap. 4 is retraced the detector s path of development, starting from the early works to the last prototype. In cap. 5 studies about the perception of the device and the tests on realistic simulations of clinical cases are represented. Results on clinical applications obtained with numerical simulation are also reported. In cap. 6 the first attempts to develop a detector with a different finality, from a counting device to an imaging probe, for a further extension of the techniques, are discussed. 1

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9 Chapter 1 The treatment of cancer The cancer s impact on population has been increasing in the last years. This has been related to different causes like the raising number of cancerogenic factors and the ageing of the population that may cause a longer exposure to them. The strong improvement of the diagnostic capabilities makes now possible to recognize, identify and correctly classify a much more wide spectrum of pathologies previously misclassified. Tomography has emerged as the most important diagnostic tool. It makes possible to achieve the clear identification of the tumour mass area allowing the doctors to make a correct and precise pianification of the operation. Today traditional surgery till remains the most applied technique in oncology, and a complete success of the surgery is fundamental for the survival patient s outcome. Unfortunately, some forms of tumours, especially those related to brain and abdomen, are very difficult to treat surgically and represent a hard challenge for the surgeons. 1.1 Cancer basics All tumours originate from a cell. In normal tissues, cells replicate and divide to satisfy the physiological growth needs of the body and the continuous turnover of dead or damaged cells [1]. In tumours, this delicate balance ruled by genes coded in DNA and run by chemical messages sent from one cell to another, is compromised. The tumoural cells continue to reproduce without restraint and the apoptosis, the physiological processes by which damaged cells undergo a programmed death, are impaired. Some of these mutations are inherited, while others may be caused by external factors as environmental factors like lifestyle (nutrition, tobacco use, physical activity, etc.), naturally exposures (ultraviolet light, radon gas, etc.), medical treatments and pollution [2]. Substances and exposures that can lead to cancer are called carcinogens. Some carcinogens do not affect DNA directly, but lead to cancer in other ways. For example, they may cause cells to divide at a faster than normal rate, which could increase the chances of DNA mutations. Carcinogens do not cause cancer in every case, all the time. Substances labelled as carcinogens may have different levels of cancer-causing potential. Some may cause cancer only after prolonged, high levels of exposure. For any particular person, the 3

10 4 1. The treatment of cancer risk of developing cancer depends on many factors, including how they are exposed to a carcinogen, the length and intensity of the exposure, and the person s genetic makeup. Today cancer is the second cause of death after cardiovascular disease, accounting for nearly 1 of every 4 deaths [3]. The best chance of cure is related to the complete removal of the entire cancer mass. The most used techniques is the surgery, alone or in combination with other cancer treatments, like chemotherapy or radiation therapy. An important margin of uncertainty is represented by the fact that even a single tumoural cell -invisible to the naked eye- remaining after the surgical removal, can develop into a new tumour. For this reason, in some cases, when applicable and in absence of metastasis, the surgeon can decide safer to remove the entire organ to prevent recurrences. The definition of a correct diagnosis requires also the correct evaluation of the extension of the tumour mass considered. In some cases, cancer cells can spread from their original point to other areas of the body through either the bloodstream or the lymph system (fig. 1.1). Lymph nodes are small, bean-shaped structures composed of immune cells, whose principal function is to filter out or kill the cancer cells. If cancer spreads through the lymph system, it usually affects the near lymph nodes. Hence, the identification and the condition of the nearest lymph node to the tumour is very important in cancer staging, which decides the treatment to be used, and determines the prognosis. Several attempts to help the surgeons in the identifications of illness nodes were made. One of these was the injection of a blue ink (such as Patent Blue V [4]) near the lesion, few minutes before the operation. The ink was then absorbed by the lymphatic vessels and in this way the node acquired a blue color, allowing an easiest identification. Due to the high percentage of failure, this method is not so far used, although the concept of marking lymph nods remains, as it will be discussed in the follow. The extension of tumour masses and their spread is defined by the staging system that uses a Roman number from I to IV, with number increase proportional to the advance and spread of the tumour. The stage is referred at the moment of the diagnosis so, even if the tumour evolves in time (and consequently the treatment) the level is fixed. The survival statistics refer to the stage at the moment of the diagnosis. Although each person s situation is different, cancers with the same stage tend to have similar outlooks and are often treated the same way, so stage information helps to formulate the plan treatment. An important part to estimate the spread of a tumour is played by the imaging tests. 1.2 Diagnostics Biomedical imaging techniques are fundamental for cancer management. Imaging forms an essential part of cancer clinical protocols and can provide morphological, structural, metabolic and functional information, allowing an inside view of the

11 1.2 Diagnostics 5 Figure 1.1. The lymph system, from American Cancer Society [5] patient from the outside. Early detection of cancer through screening based on imaging is probably the major contributor to a reduction in mortality for certain cancers [6] Computed tomography Since its introduction in 1971, Computed Tomography (CT) has become an important tool in medical imaging [7]. This techniques combines many X-ray images taken from different angles to produce an image of the scanned object. X-ray imaging is based on the different absorption of X-rays in function of density. Quantifying the intensity change of X-rays that exit the body can provide an internal view of the patient using the density diversity among the different organs (i.e. adipose tissue has a density of 0.9 g/ml, while muscular tissue has a density of 1.06 g/ml). To increase the differences, an injection of a contrast substance, such as intravenous iodinated, can be used to facilitate the identification of the blood vessels otherwise difficult to delineate. A 2-dimensional projection image of the internal of the patient s body is called radiography. Conventional medical CT instruments are able to provide radiography with a resolution on the order of 1-2 mm. An example is reported in fig The drawback of this type of imaging is that the amount of X-rays absorbed can cause damage to the patient leading to cancer (with a probability of 0.1 % [8]).

12 6 1. The treatment of cancer Figure 1.2. Cross-sectional CT image of abdomen Magnetic resonance imaging Another medical imaging technique widely used today is Magnetic Resonance Imaging (MRI), utilized to investigate the anatomy and the physiology of the body [9]. MRI involves the use of a magnetic field gradient across the tissue. In this way a given value of the field is associated with a specific body location. The magnetic field partially polarizes the nuclear spin of the proton, while a radio frequency is used to excite the spin. Since the frequency of the signal emitted by the protons during their disexcitation is proportional to the magnetic field, this allows to reconstruct a map of the tissue as a function of the number of protons present. Many of those protons are the protons in water (hydrogens), so MRI is particularly well suited for the imaging of soft tissue. MRI is able to provide an excellent contrast and images with high resolution (order of 1 mm), allowing to discriminate between organs with similar density like the spleen and the liver. This diagnosis does not involve the use of ionizing radiations, so its use is generally preferred to other techniques, information being equal, even if it is more expensive and time consuming. Figure 1.3. Identification of a brain tumour with MRI

13 1.2 Diagnostics Nuclear Imaging (PET and SPECT) Nuclear imaging uses low doses of radioactive substances linked to compounds that act bounding to tumour cells (as will be detailed in sec. 2.1). Using scintillator detectors, it is possible to identify the areas that show an higher radioactivity, localizing consequently the tumour masses. The two major instruments of nuclear imaging are Positron Emission Tomography (PET [10]) and Single-Photon Emission Computed Tomography (SPECT [11]) scanners. A PET scans is finalized to the creation of computerized images of chemical changes, such as sugar metabolism, that take place in the body. Typically, the patient is injected intravenously with a radioactively labelled sugar (usually fluorodeoxyglucose - FDG - marked with 18 F ). Cancer cells are constantly reproducing, thus need a lot of energy in the form of glucose molecules. FDG administered to the patient is preferentially absorbed by tumoural cells, making this molecule an efficient carrier towards the cells cancer due to its metabolic pathways. In a PET scan the tracer coupled with the carrier is a positron emitter ( β + decay). Positrons travel through human tissue giving up their kinetic energy principally by Coulomb interactions. When they reach thermal energies mainly interact with electrons by annihilation, producing two 511 kev photons. With the reconstruction of the positron s annihilation point, it is possible to reconstruct the images of the tumour masses, where a higher numbers of positrons are expected to annihilate. PET imaging is subject to intrinsic limitations in spatial and temporal resolution resulting from the physics of positron annihilation, the imaging technique, and statistics. The annihilation radiation comes from the positron stopping point, located at some distance from the decaying nucleus. For the energy of interest, in the order of one MeV, this distance is about 2-3 mm. Also the two γ rays that result from positron annihilation in atoms are not exactly collinear because atomic electrons are not at rest and this could cause inaccuracies in the reconstruction of the event. The statistical noise in PET images is determined by the number of image counts, which is dependent on the tracer and isotope, the administered activity (limited by radiation exposure considerations, usually in the range 2 to 15 MBq/kg [12]), the scan time and the scanner efficiency. Also it is determined by the algorithm of images reconstruction from the projections acquired. At best, a reconstructed image has several million reconstructed counts. A scan takes approximately minutes, depending on the type of scan planned and the type of scanner being used. PET shows metabolism change and may plays a fundamental role in determining whether a mass is cancerous. PET scans may detect cancer when other imaging techniques show normal results due to the fact that the biochemical lesion occurs before the physical damage. However PET is more accurate in detecting larger and more aggressive tumours since it can not locate tumours smaller than 1 mm 3. PET can be used to check if a treatment is working (cancer cells are dying) detecting the return to a normal sugar metabolism. A similar technique is the SPECT, with the difference that this techniques uses

14 8 1. The treatment of cancer tracers that decay producing a photon, used for image reconstruction. Generally SPECT radio tracers last longer in the patient and are primarily applied in cardiology where myocardial stress imaging takes about three to four hours. Figure 1.4. Images of all body scans using SPECT (left) or PET (right) 1.3 Intraoperative localization of cancer cells As aforementioned, the goal of a cancer treatment is the complete and precise resection of the lesion. There is a strict correlation between patient survival, disease free time and the resection completeness. The translation of pre-operative imaging information in the surgical field is a critical point to improve the surgery outcome, minimizing the surgical invasiveness thus maximizing benefit to the patient. However during this translation, problems could occur. Standing the low resolution of the PET images, a small volume detected as potentially tumorous could not be easily identifiable in the patient. In addiction tumour massed could slightly move their position during the surgery, as typically happens in brain surgery, where after craniotomy the brain collapses. In this way, the precise identification of tumour remnants after the resection of the bulk mass can result difficult for the surgeon. A way to effectively help the surgeons to identify tumoral tissue during the intervention, less reliant from pre operative images, is to inject the patient with a cancer cell probe, that is a molecule that somehow bounds to the neoplastic cells, and the detection of which is possible by a detector Surgical navigation Computer Assisted Surgery (CAS) represents a set of methods that use computer technology before and during the surgical intervention. For the use of this techniques is important to develop a model of the patient, reconstructed from medical imaging technologies. The objective is the creation of a 3D model that exactly reproduces the structure and the pathological tissue of the area of interest. Thanks to this virtual model of the patient, surgeons can establish a better plan of intervention, because it can be simulate before the surgery.

15 1.3 Intraoperative localization of cancer cells 9 In CAS the actual intervention is defined as surgical navigation. Using a surgical navigation system, the surgeons use special instruments tracked by the navigation system. The position of the instruments is shown in real time on a model of the patient, allowing the reach the area of interest without identification problems. The system s feedback about instrument location is particularly useful in minimally invasive surgeries, where the operators can not see the tip of the instruments. CAS allows to test the operation before surgery, and this train reduces the risk of surgical errors and the operating time. A major disadvantage of this system is the high cost, of the order of a million e (100 ke/y the cost of maintenance). The size of the system may represents an important limit of application in operating theater (fig. 1.5). Figure 1.5. Size of a typical CAS robotic equipment (da Vinci surgical system) Fluorescence-guided surgery Fluorescence-Guided Surgery (FGS) is a way to identify tumoural tissue during the intervention [13], using fluorescence light (photons in the Near Infrared -NIR- with a spectrum between nm) emitted by a molecule that bounds to a cancerous mass. This phenomenon allows an intraoperative visualization of the area to aid the surgeon with a real-time tumour identification and delineation making possible a precise removal of cancerous tissue. A dedicated camera system has to be used during the operation to detect the fluorescence light emission, being the human eye not sensitive to light in the NIR region. FGS is cheaper compared to other medical imaging techniques, and allows a better resolution. However, detection depth limitation exists. In the visible light region, tissue absorption limits the penetration depth to a few millimeters with a maximum of 10 millimeters [14]. In addition, these procedure is quite unhandy, since there is the necessity to shift between normal light and fluorescence light, and this means to repeatedly switch off environment illumination.

16 10 1. The treatment of cancer Figure 1.6. Identification of a neoplastic mass with the XRF techniques. The same area is shown with normal light and infrared light. The green light easily allow to localized the mass -from da Vinci promotional video 1.4 Radioguided surgery Another techniques aimed to identify tumoral tissue during the intervention is the RadioGuided Surgery (RGS). RGS is a surgical technique that enables the surgeons, during the operation, to identify malignant tissue marked before surgery with a radionuclide. Essentially RGS consists in a compound that is able to bound with the tumour and carries a radioisotope, the radioisotope and a detector. A scheme of the techniques is shown in fig Two different approaches to RGS, one with gamma and one with β that will be discussed in the follow are also shown. The compound, or carrier, must be taken up by the tumour far more with respect to the surrounding healthy tissue. The metabolism (uptake and wash out) of the compound has to be consistent with the requirements of the surgery and the half-life of the isotope. The chemical proprieties of the radioisotope must allow it to form a stable labelled compound. The radioisotope must have an appropriate half-life and emits particles compatible with patient safety and detector system. The detector system has to be handly to allow a minimal invasive surgery, it has to respect the guidelines of surgical environment, and must give an easy to comprehend feedback. The concept of RGS began in the 50 [16]. In the years following World War II ( ), several investigators [17] [18] realized that if one could marker a tumour by making an intravenous injection with a compound labelled with a radioactive isotope it could be possible to locate the tumour scanning the region under suspicion with a radiation detector. In these early works, the isotope chosen was 32 P (14.3 days half-life, 1.71 MeV β ) and the detector was a Geiger-Müller tube (GM) working in avalanche mode. GM was a simple ionization chambers, filled with an argon-ether mixture, that collected the ions produced in the gas by the incident radiation (fig. 1.8). GM was chose since it was one of the first detector developed. It could be useful remind that particles detection was moving its first steps, so there were not all the possibilities available today.

17 1.4 Radioguided surgery 11 Figure 1.7. RGS procedure: (1) before the operation, the patient is injected with a radiolabelled tracer; (2) the emitting tracer is preferentially taken up by the tumour; (3) after the cancerous bulk removal, the surgeon explores the lesion with a radiation detecting probe looking for targeted tumour residuals in real time. The bottom boxes show the differences about γ emitting tracers (a) and electron emitting tracers (b). Due to the high penetration power of the photons, in the first case a non-negligible background can be produced by the healthy organs close to the lesion, sometimes preventing the applicability of the technique. To mitigate this effect a shielding or active veto has to be applied (see inset of box a). In case of electrons, the effect of background is reduced, providing a clearest delineation of radioactive tissue s margins and allowing for a simple and compact probe [15] Figure 1.8. Photograph of the earliest GM tubes used as intraoperative probes. (From [19]) The techniques was phased out due to problems both with the radiotracers and the detector. The disadvantage of 32 P was that, considering the low uptake, to obtain enough statistics the dose injected was MBq (1-4 mci), 100 times the dose that today is normally accepted in nuclear medicine. Also inject a patient with an isotope with half-life of 14 days caused as secondary consequences the radioactivity of the patient for a long time. It has to be noticed that medical knowledge about effects of radioactive isotopes on the patients was scarce in those years. Furthermore the massive detector not easy to handle, the need of HV, and the necessity of gas, that is potentially explosive, put several limits on the possibility of a clinical use.

18 12 1. The treatment of cancer In the 1950 s a new γ-emitting isotope the 99m T c started to be used. 99m T c due to its pure photon without beta contamination emission and its short half life of 6 h, gained wide consensus and till today is the most used radionuclide. New radiotracers marked with this isotope were developed, allowing to overcome some of the previous limits and the technique reborn, replacing tracer marked with β emitters with γ emitters. As consequence, the studies of β -probe were abandoned in favour of γ-probe. RGS with γ has to deal with the high penetration power of gamma rays. This implies that background events could come from any part of the patient, and since it is rare to have more than a few percent of the isotope in the tumour, the total background from the rest of the body can be very significant. The early scintillation detectors (essentially a crystal coupled to a light sensor) were shielded by various amounts of platinum to improve the directionality of the field of view. In the mid 1980s, several companies started to offer basic γ-detecting intraoperative probes and their were normally used in hospital. In RGS there are several approaches regarding the way to carry the tracker compound to the tumour [20]. The approaches could be divided in two main areas, if they look or not for lymph node. As said, the tumour cells can spread across the blood or across the lymphatic vessels. The spreading in blood is impossible to control, differently from that in the lymph, due to its slower circulation. This fact is exploited in Sentinel Lymph Node Localization (S.L.N.L.). The hypothetical first lymph node or group of nodes draining a cancer is referred to as sentinel, exactly upon the assumption that in case of dissemination this is the target organ primarily reached by metastasis cancer cells from the tumour. It is thus fundamental to inspect this lymph node for the staging of the cancer, subjecting it to biopsy and histologic analysis. Today for S.L.N.L. there is consensus on the use of Technetium-99-labeled colloids with well defined properties in terms of particle size (about 100 nm). The tracer is inject close to the tumour, and then the physician uses a particle detector to search for activity of hot spots in order to identify the sentinel lymph node. The group of nonsentinel lymph node applications of RGS can be in turn roughly divided into 2 main categories, according to the kind of radiopharmaceutical. Some are based on radio-pharmaceuticals that do not have any tumour-seeking property, but are nonspecific particulate agents that do not appreciably move from the site of injection, that is local with respect to the tumour. It is a mechanism similar to that of S.L.N.L., but with the size of the molecules changing on the basis of the needed retention time of the colloid within the cancer. On the other hand, there exist radiopharmaceuticals that accumulate preferentially at tumour sites, and are thus administrated systemically [16]. RGS brought a relevant improvement in breast cancer surgery. In this case, as in others, an early detection decreases mortality, but early-detected tumours are generally small and non-palpable. Thus often it is difficult for the surgeon to find

19 1.4 Radioguided surgery 13 them intraoperatively even if they are clearly identified before the operation. In this context the Radio-guided Occult Lesion Localization (ROLL) technique has been proposed. A small dose of colloid marked with 99m T c is administer near the lesion, allowing to remove the tumours even if invisible by naked eyes. This technique, first proposed in 1999, is nowaday wide common in hospitals, and has been proven to improve patients outcome [21]. RGS with γ is considered an established discipline within the practice of surgery. Current application of radioguided surgery are colon cancer, sentinel-node mapping for malignant melanoma and breast. An extension of the techniques has been proposed also for recurrence of renal cell carcinoma, cervical recurrence of papillary carcinoma of the thyroid, recurrence of retroperitoneal sarcoma, lesions of the popliteal fossa, and neuroendocrine tumours [22] [23] [24]. Today all the RGS applications need a specific probe able to detect photons of energy of about kev. The rapid diffusion experienced by RGS in the last decades resulted in a proliferation of commercially available instruments for this purpose, like the one in fig These probes are usually composed by an active area (scintillator or semiconductor detector), surrounded by few millimeters of shielding material, in order to retain directionality. The instrument is then connected either via cable or wireless to an electronics apparatus for signal elaboration. The information to evaluate the activity measured are given to the surgeon usually by means of different feedback (small display, color/sound code). Usually probes are counting devices, but more recently intraoperative imaging probes have been developed. These devices add the ability to see the details of the detected activity, giving the potential of using the technique in a low-contrast environment. The systemic administration of the radio tracker results in a relatively high radioactivity concentration in normal tissues surrounding the tumour, depending on the affinity of the used molecule. Considering the high penetration power of the photons, even an uptake of tracers far from the lesion, due to the metabolism, can increase the amount of background. This affects the signal to noise ratio, where signal is defined as the rate over the tumour. To increase this ratio, the probe have to be shielded increasing the traverse section of the probe, making it not so handy for the use in a narrow and complex operating field. Nevertheless in particular areas of the body, like the abdomen, it is not sufficient. In fact the presence of the spleen and the liver, that have an high uptake of radiotracer for pure metabolism, prevents the use of RGS in this area, since they act like a "lighthouse" hiding the signal from the lesion. Lastly photons have enough energy to escape the body patient, causing a non negligible exposition for the medical personnel during the surgery. Dosimetry has to be taken in account. Since today the main drawback of this technique is related to the choice of the

20 14 1. The treatment of cancer Figure 1.9. Example of commercial available gamma probe (Neoprobe 2100). The probe is wired to a box to display the counts acquired. Different models are available with Bluetooth connection instead than wire photons, a development changing the emitting isotope has been proposed Use of β+ decay The first proposed alternative was the use of radionuclides that decay β +. The choice to detect positrons has several advantages. Positrons have a cross section similar to that of the electrons (fig. 1.10) and, as shown in fig. 1.11, their range in water is lower than photons. The penetration depth of the cm allows a better signal to noise ratio, because the radiotracers uptake far from the lesion can not contribute background. Lastly all the emitting isotopes that are already used for PET imaging could be used. PET tracers are well know and there are established protocols. Since the identification, creation, testing and legislative permission to bring a new radiotracer into clinical tests is a very long and expensive path, this opens - in a quite easy way - the possibility to extend the techniques to a lot of new cases. In 1994 a β + probe was proposed [27]. The probe consisted of a groups of plastic scintillating fibres packed annular (fig. 1.12). Each detection fibre was associated with a close fibre encapsulated in a thin foil of stainless steel. In this way the probe worked like a two-detectors system. The shielded fibres counted the photons, while the others both positrons and photons. In this way the number of positrons was obtained subtracting the activity measured by the photon counting fibres to the positron counting fibres, thus eliminating the fraction of photons acquired by the positron detector.

21 1.4 Radioguided surgery 15 Figure Comparison between the cross section of electron and positron in lead from [25] Figure Range in water for different particles from [26] Figure The prototype of β + probe proposed by Bonzom et al., coupled with an excision tool commonly used in neurosurgery

22 16 1. The treatment of cancer The effect of γs contamination was crucial in β + probe, and was the main limit of the techniques. The annihilation caused a non negligible background. Photons with 511 kev had an high penetration power, (β + tracer are tough for diagnostic, γ have to interact outside the patient s body) so the situation was similar to γ-rgs. The dual probe mode tried to fix this problem, but the consequence was the creation of a larger detector (actually two probes, one for photons and another for positrons are needed). In addition, an important complication was represented by the fact that to acquire enough statistic to make an on line background subtraction, it was necessary to increase the dose delivered to the patient (with obvious exposure problems for the patient and for the medical personnel) or to wait a longer integration time increasing the overall duration of the surgery. In 2007 this probe was proposed by Bogalhs et al. for β + -RGS in brain surgery [28] and was tested on 18 F phantoms. They succeeded to detect phantoms of 5 mm in 5 s, but this promising result was no further developed Use of β- decay Considering the innovations and the developments of medicine and nuclear physics, we thought possible to overcome the drawbacks of β RGS, that, although was the first approach to radioguided surgery is practically unused today. The main advantage of β surgery compared to the traditional RGS using γ radiation is a more favourable ratio between the signal coming from the tumour and the rest of the body. Conversely to the tracers with γ emitters, pure β radionuclides emit electrons which penetrate only a few millimetres of tissue and produce almost no gamma radiation (probability of Bremsstrahlung contribution less than 0.1%) resulting in a very low background on the lesion signal. Operating in a low background environment allows the development of a handy and compact probe which, detecting particles emitted locally, provides a clearer delineation of margins of the lesioned tissue. The RGS technique applicability can therefore be extended also to cases with large uptake of the tracer from healthy organs next to the lesion, minimizing the radiotracer activity to be administered. Lower exposure of the medical team is also expected. My Thesis work was focussed in the translation of this proposal into a set of measures to verify its applicability. This means identify relevant clinical cases, suitable radiotracers and the development and testing of a beta probe, in a way as close as possible to the surgical environment.

23 Chapter 2 Medical applications As shown in section 1.4, although γ-rgs is a powerful tool, it has several limits that could be overcome moving to β -RGS. To verify this hypothesis, the availability of suitable tracers capable of delivering pure β emitting radionuclides is necessary. In this chapter there will be a general overview about radiotracers, then the discussion will be focussed on the DOTATOC marked with 90 Y, the tracer chosen for first applications of β -RGS. Studies of uptake estimation on possible clinical cases of interest are also reported. 2.1 Radiotracers A generic drug used for medical purpose in which there is the presence of radionuclides is defined as radiotracer. These drugs have the ability to temporally transform the tissue in a radiation source, thus can be applied to localize the target tissue (in diagnostic) or to try to kill them (in therapy). The basis for the application of radionuclides in medicine comes from the works of George de Hevesy that was a pioneer in the field of tracers, since he was the first to use them as tracers to follow the distribution of marked compounds into plants [29]. The invention of cyclotron in 1932 (the first cyclotron for medical purpose was built in 1941) allowed the production of synthetic isotopes increasing the number of potential radiotracers. Starting from then, the utility of radiotracers in nuclear medicine has been expanding continually till today. Radiotracers consist of two parts: a carrier and a radioactive isotope. The carrier is a compound able to bound both with the isotope and the tissue of interest, while the isotope, through its decay, allows to track the carrier. The choice of the isotope is driven by the possibility to replace an atom of the carrier without changing its metabolism, and must take into consideration the decay time and the type of decay. Moreover it must not change the pharmacokinetics of the drug. 17

24 18 2. Medical applications Radioactive isotopes The radioactive isotopes can emit different ionising radiations. The decays of interest, in the field of medicine, are γ, α and β. The type of emission and its interaction with the matter decide the field of application of the isotope Gamma decay Usually, gamma emitting isotopes used in nuclear medicine release a photon in an energy range between 50 and 600 kev. A table of the most used gamma radiotracers is reported in 2.1. In this energy range the photon can be completely absorbed (Photoelectric effect) or can scatter with an electron (Compton scattering), being the pair production denied since requires a photon s energy higher than MeV (in fig. 2.1 are shown the cross sections of the different processes as a function of photon s energy). Radionuclide T 1/2 Photon s energy [kev] Use 99m T c 6.01 h 140,142 Scintigraphy, liver function 51 Cr 27.7 d 320 Diagnosis of gastrointestinal bleeding 57 Co 272 d 14,122,136 Radiolabel for vitamin B 12 uptake 67 Ga 3.26 d 94,184,296 Tumour imaging 123 I 13.2 h 159 Diagnosis of thyroid function 125 I d 35 Cancer brachytherapy 201 T l 3.04 d 71, 135, 167 Diagnosis of heart conditions Table 2.1. Some of the γ emitting tracer used in nuclear medicine. For each isotope is reported the half life, the photon s energy and the field of application The combined cross section of gamma rays is much smaller than cross sections for charged particles undergoing inelastic electron collisions (in the same energy range), as consequence, photons have a higher penetration power. Considering their free mean path, shows in 2.2, photon have a high probability to escape from the patient s body. For this reason are mainly used for imaging purpose (i.e. SPECT). The radioisotope most widely used in medicine is an artificially-produced element, the Technetium-99metastable, employed in some 80% of all nuclear medicine procedures ( 30 million per year). 99m T c has a half-life of six hours, short enough to minimise the radiation dose to the patient, and at the same time, long enough to be consistent with the metabolic processes. It decays by an internal transition to 99g T c (t 1/2 =0.2 million years) that is so long-lived that it can be regarded as stable. The low energy gamma rays of 141 kev emitted by 99m T c have the possibility to exit the human body and to be

25 2.1 Radiotracers 19 detected by a gamma camera (approximately 5 cm of tissue between the radionuclide and the detector stop about half of the photons whereas 10 cm stop about three quarters). In addition the chemistry of technetium is so versatile that a wide range of substances can marked with it, increasing its field of application. Finally, Technetium-99m generators, device used to extract 99m Tc from a source of 99 Mo, are available. These generators allow to extract the isotope directly into the hospital, avoiding problems related to the transport of isotope with "short" half life. Figure 2.1. Photon total cross sections as a function of energy in lead, showing the contributions of different processes (from [25]): σ p.e. =Photoelectric effect σ Compton =Compton scattering k x =Pair production Figure 2.2. Photon free mean path λ for various element as function of photon energy. For a chemical λ Z mixture, λ eff Σ elements, w Z where w Z is the proportion by weight of the element with atomic number Z (from [25]) Given these favourable properties of 99m T c, over the time a multitude of technetium compounds has been developed [30], as example: 99m T c-phosphates and diphosphonates are used for bone imaging 99m T c-sestamibi and 99m T c-tetrofosmin are used to measure myocardial blood flow during a stress test and at rest, allowing diagnoses of myocardial infarcts and coronary artery disease 99m T c chemically bound to exametazime (HMPAO) is used to detect cerebrovascular diseases. This radiopharmaceutical, Ceretec TM, is also used to label white blood cells to localized infections. Today there are more than 30 radiopharmaceuticals based on 99m T c for imaging and functional studies of brain, myocardium, thyroid, lungs, liver, gallbladder, kidneys, skeleton and blood Alpha decay This is a modality of decay for unstable heavy atoms with at least an atomic number of 82. Alpha particle has a high ionizing power being quite massive and having a double positive charge. For this reason its penetration depth is very low (less than 1 mm in water). In this way, luckily, alpha particles are not able to penetrate the outer layers of skin, so in general, are not dangerous unless the source is ingested or

26 20 2. Medical applications inhaled. These characteristics make this radiation very suitable for internal therapy. Different carriers able to deliver alpha emitters into cancerous cells were developed. In this case, the damage to healthy tissue is limited to the short range of interaction. It must be emphasized that the higher ionization power increases the ability to cause biological damage, causing a direct break of the double-strand DNA, which leads to cancer cell apoptosis [31]. A given amount of energy absorbed in the form of alpha particle should be assumed to produce 20 times the damage caused by an equal amount of energy absorbed as gamma rays. Today Xofigo ( 223 Ra dichloride) is used as a cancer treatment for bone metastasis. 223 Ra has an half life of 11.4 days, and produces (considering its chain of decay) alpha particles in the energy range from 5 to 7.5 MeV Beta decay Isotopes that undergo a beta decay can be used both for diagnosis and therapy. For diagnosis are used β + decay. The positrons emitted have not enough energy to escape from the patient and annihilate near the decay point, producing two 511 kev γs. The image is then reconstructed studying the position of the annihilation points (as discussed in section 1.2.3). In case of β the electrons path is short enough to deliver the dose to the target tumour cells (the same principle of the use of alpha decay), so this type of decay is used for therapy. Several positron-emitting radioisotopes are used for PET imaging (some of them are listed in tab. 2.2). This abundance has lead to the creation of a vast array of biological radiotracers with a high importance in nuclear medicine. Today 18 F -FDG (fluorine-18 labeled 2-flouro-2-deoxy-D-glucose) is the PET most used radiotracer. In general, malignant cells tend to consume glucose in preference to free fatty acids. In addition, cancer cells tend to use an anaerobic metabolism, that requires much more glucose than the aerobic one. FDG imaging reflects glucose metabolic rate, so it suits well for cancer imaging [32]. The positron emitters 11 C, 13 N, and 15 O are not used commonly in clinical practice primarily because of their short half life. Anyway 11 C can be used for blood volume determinations, ammonia tagged with 13 N is used to myocardial perfusion imaging, and 15 O gaseous is used as metabolic agent [33]. Although radiotherapy is a less common use of radioactive materials in medicine, it is nevertheless important. β emitters well suit this use. Beta particles normally used are in the energy range from 0.05 to 2.3 MeV. A list of β emitting isotopes with the energy of beta decay is reported in tab. 2.3.

27 2.1 Radiotracers 21 Radionuclide T 1/2 Average Positron s energy [kev] 11 C 20.4 m N 10 m O 2 m F 110 m K 7.6 m Cu 9.7 m Ga 68.1 h 836 Table 2.2. Some of the β + emitting tracers used in nuclear medicine. For each isotope is reported the half life and mean value of the positron s energy The path of electrons in tissue is of the order of a centimetre, comparable with larger size tumours. Electron ionization power is lower than alpha, so higher radionuclide concentrations compared to alpha emitters are required for equivalent rate of cells kill. However the path of electrons allows them to cross multiple individual cells, decreasing the need to target each cancer cell with a radionuclide emitter. Today the importance of β radiation arise mainly from treatments with 131 I and 90 Y. Large administered activities of 131 I are commonly used for treatment of hyperthyroidism and thyroid cancer. Although 131 I is a beta emitter, there is a predominant energetic gamma emission (364 kev), which can be used to study the tracer distribution. Particular radiation protections have to be taken in consideration for the patients after this treatment, since this gamma emission could result in a measurable absorbed doses to people near the patient. 131 I is also used linked to tositumomab (Bexxar ; GlaxoSmithKline, Philadelphia) for non-hodgkin s lymphoma as the 90 Y ibritumomab tiuxetan (Zevalin ; Biogen Idec Inc., Cambridge, MA [34]). 90 Y as marker for a somatostatine analogue, DOTATOC, is used for peptide receptor radionuclide therapy. Radionuclide T 1/2 Emission [MeV] 32 P 14.3 d 1.71 max; 0.7 mean 89 S 50.5 d 1.46 max; 0.58 mean 90 Y 64 h 2.2 max; 0.93 mean 131 I 8 d 0.6 max; 0.19 mean; γ 364 kev (82%) 153 K 46 h 0.81 max; 0.23 mean; γ 103 kev (28%) 186 Re 90 h 0.76 max; 0.34 mean; γ 186 kev (9%) Table 2.3. Some of the β emitting tracers used in nuclear medicine. For each isotope is reported the half life, the mean and maximum value of electron s energy and, when appreciable, the rate of the principal gamma emission

28 22 2. Medical applications Peptide receptor radionuclide therapy (PRRT) is a molecular therapy used to treat neuroendocrine tumours (NETs). This type of tumour is related to endocrine and nervous systems. A possible extension to prostate and pancreatic tumours is under investigation. Most neuroendocrine tumours present an abundance (overexpression) of a specific type of receptor, the SSTR 2, a protein that extends from the cell s surface and can bind to the hormone somatostatin. Octreotide is a laboratory made version of this hormone with the same property of binding to somatostatin receptors in neuroendocrine tumours. Octreotide marked with a radionuclide creates a radiopeptide, a type of radiotracer. When injected into the patient, these radiopeptides travel into the bloodstream and reach the site of neuroendocrine tumour where there are uptaken by the receptors. 90 Y and 177 Lu are the most commonly used isotopes to mark octreotide. The radiopeptides are highly selective so the radiation exposure of healthy tissues is limited. As result, PRRT typically has milder side effects compared with chemotherapy [35]. The purpose of PRRT is not curative, as therapy is used to relieve symptoms and slow or stop the progression of the disease, improving overall survival. Individuals whose tumours can be visualized by somatostatin receptor scintigraphy or 68 Ga-DOTATE PET/CT and have inoperable growing tumours may be candidates for PRRT Carriers The function of the carrier is to transport the radioisotope to the tumour site. It must be able to bound both to the isotope and the tumour cells. Several criteria need to be evaluated to find the isotope s suitable carrier [36]. The tumour s biology and the anatomic location in the body, its shape and size, the presence or not of direct access routes to tumours such as vasculature or direct administration in the space where they may be confined (blood or peritoneal cavity) are aspects that play an important role in this selection process. Also the choice depends on the target affinity and stability of the ligand-labeled carriers, and their resistance to degradation due to radiation. Stable radiolabeled constructs are crucial, since released radionuclides are free to distribute throughout the body and potentially accumulate in normal organs thus increasing toxicity. The dose limiting toxicity is the major limitation in internal cancer therapy. The accumulation of radiotracers in organs other than the target one, prohibits the administration of higher doses that could reach lethal absorbed levels at the tumour sites. The radionuclide half-life and body s washout are parameters used to optimize the value of the injected dose. To result in high tumour absorbed doses with relative low normal organ accumulation, a variety of designs for targeting carriers combined with different radionuclides are proposed. Essentially there are two type of targeting: active and passive.

29 2.2 Radiotracer for β -RGS 23 Active targeting is also called ligand based targeting. In this process there is the use of biological processes to allow a drug accumulation in the target cells, attaching to the drug something like a antibody able to bound to cells receptors. In passive targeting, the physicochemical properties of the drug carrier complex are modified, so that it escapes body defence systems and accumulates in the target tissue. Drug delivery systems that have been developed to be used as passive targeting carriers include micelle, nanoparticles, polymeric conjugates and liposomes. Considerable medical research is being conducted worldwide into the use of radionuclides attached to highly specific biological chemicals such as immunoglobulin molecules (monoclonal antibodies [33]). Antibodies are selective for receptors that are overexpressed on a certain disease site such as tumour cells. In radioummunotherapy their are used to treat lymphomas. 2.2 Radiotracer for β -RGS Find a tracer suitable for β -RGS is mandatory to verify the proposal. Some are the characteristics required about the isotope: - Clear beta decay without emission of photons, or very low rate of photon emission, in order to reduce the background - Electron s mean path in the order of mm, resulting in an endpoint neither too energetic nor too low, to find a balance between the probe identification power (discovery power and sensitivity to depth) and the background rate - Practical half life, enough to be consistent with the tracer s uptake, but not too long to be compatible with patient s radio-protection The development of a new radiotracer is a lengthy (8-10 years) and costly ( million) process, mainly due the clinical trials, and, in addiction, despite these heavy investments, there is a high rate of failure [37]. Considering this, the development of a new radiotracer with all the desired characteristic required for a β -RGS was not a viable path, especially as first application. To test our proposal, we had to find a suitable radiotracer among the existing one. 90 Y -DOTATOC, a beta tracer used in PRRT, was identified as the best candidate for the first trails. The carrier of this tracer is the (DOT A 0 P he 1 T yr 3 )octreotide, or DOTATOC, a somatostatin analogue used for the treatment of neuroendocrine tumours, whereas the receptors for this hormone were identified also in tumours of the central nervous system, breast, lung and lymphatic tissue [38]. The carrier is marked with 90 Y, an isotope which decays in 90 Zr through a pure β decay (99.9 % of the time, as shown in fig. 2.3). The electron s endpoint is 2.28 MeV, meaning a maximum path in the body of 11 mm. An half life of 64 hours

30 24 2. Medical applications completes the decay scheme. It must be considered that even if the isotope is a pure beta emitter, there is always the possibility of Bremsstrahlung radiation. While this possibility is exploited in PRRT to acquire a low resolution image for the validation of the treatment, in case of RGS it could represent a drawback. However, since the probability of this process is low (0.1 %) and the photon s energy spectrum is peaked at 100 kev (as shown in fig. 2.4), this aspect is expected to be neglectable. Figure Y decay scheme Figure 2.4. Simulated Bremsstrahlung s energy spectrum in human body generated by 90 Y decay 2.3 Clinical cases of interest The presence of an existing radiotracer marked with a promising isotope is a condition necessary but not sufficient to start to use it. There will be the necessity of RGS for the type of tumours the tracer was thought. This is the case. Neurosurgery is one of the fields that would profit most from RGS, given the importance of complete lesion removal being at the same time conservative to preserve neurologic functions. Presurgical imaging is scarcely effective in the search of small tumour masses because the operative field changes substantially during the resection of brain tumour. The meningiomas uptake of DOTATOC is known, and recent studies show that also high-grade gliomas present somatostatin receptors. Considering all the aspects, 90 Y -DOTATOC is an optimal candidate tracer for a first application of RGS with β radionuclides in brain tumours Brain tumours The brain is the most complex organ of the human s body. Usually a brain has a volume between 1100 and 1200 cm 3 (slightly differences between men and women). It is located in the head and protected by the skull. Between bones and brain the are the meninges, membranes that envelop the brain. The meninges consist of three layers (fig. 2.5) the dura mater, the arachnoid mater, and the pia mater, that act as defence for the brain.

31 2.3 Clinical cases of interest 25 The cerebral cortex is divided along the sagittal plane into two zones, the right and the left hemispheres, nearly symmetrical. Each hemisphere is conventionally divided into four lobes, the frontal lobe, parietal lobe, occipital lobe, and temporal lobe. Figure 2.5. The meninges: dura mater, arachnoid mater and pia mater The most common primary brain tumours are Meningiomas and Gliomas. Meningiomas originate from the meninges, the membranous layers surrounding the central nervous system. They arise from the arachnoid cap cells of the arachnoid villi in the meninges. These tumours usually are benign in nature, with a low growth. However, a small percentage is malignant [39]. A benign tumour could be completely cured if entirely removed, otherwise recrudescence could occur, even many years after the first treatment. In tab. 2.4 are reported the probabilities of recurrency as a function of meningiomas grade. Due to the particularity of the brain, even a benign tumour has to be removed. In fact the volume of brain is fixed by the skull, and any abnormal tissue growth causes a pressure on brain tissues, changing their behaviour. The annual incidence of meningiomas is 3-4 / people. Tumour Type Incidence Recurrency 3-y 5-y 10-y survival survival survival Benign (grade I) 90 % 0 % 86 % 74 % 67 % Atypical (grade II) 7 % 41% 67 % 58% 33% Anaplastic (grade III) 3 % 75 % 33 % 8 % 0% Table 2.4. Are reported the recurrency probability and the outcome of meningiomas as a function of the time. The stats are divided as a function of the meningiomas grade. The incidence of each grade is also reported [40] Glioma is a type of tumour that starts from the glial cells, the cells that surround and support neurons in the brain and that represent about half of the brain s volume.

32 26 2. Medical applications Usually they appear as malignant tumours. Surgery remains the standard of care, but the treatment can involve also chemotherapy and radiotherapy. Median survival with standardized therapy involving chemotherapy with temozolomide is 15 months. Median survival without treatment is less than 5 months. The annual incidence of gliomas is 6-7 / people. Glioblastoma multiforme, also known as glioblastoma is the most aggressive malignant primary brain tumour. It is a highly vascular tumour and has tendency to infiltrate. About 50% of the people diagnosed with this disease die within one year, while 90% within three years. Recurrency appears in 50% of cases within 8-9 months. It is also the most difficult to cure, being little sensitive to both chemotherapy and radiotherapy, and due infiltrations that make difficult a surgical resection. Anyway, despite the difficulties, surgery remains the first line of therapy. Surgery consists in a craniotomy in which a section of the skull is temporally removed to access to the brain (fig. 2.6). Then the dura is cut with surgical scissors and folded back to expose the brain. The skull tightly encloses the brain and tissues cannot be easily moved aside, making difficult this type of operation run with the help of very small tools. Sometimes the removal of other healthy tissue, like the dura, can be necessary depending on tumour s nature and its position. As a consequence, surgery can eventually comport several kind of damages, as loss of mental functions or senses, and sometimes lead to permanent disabilities. Figure 2.6. A portion of the bone is cut with a special saw and removed, then the dura is opened to expose the brain (from [41]) Early stage of meningiomas can usually be surgically resected achieving a permanent cure. Total removal is nearly impossible for higher grades. Gliomas also are resected with surgery, to relieve pressure in the brain caused by the tumour, but due to their infiltrative nature, usually this attempt are not able to cure the patient.

33 2.3 Clinical cases of interest Uptake evaluation: meningiomas and gliomas A fundamental parameter to estimate is the radiotracer s uptake. This could be tricky since the uptake of 90Y-DOTATOC can not be evaluated directly. Electrons have not enough energy to escape outside from the patient, making impossible to acquire a map of tracer s distribution. To overcome this problem, the uptake was evaluated with PET scans after the administration of 68Ga-DOTATOC, under the reasonable assumption that the uptake and the pharmacokinetics of the drug depend only from the carrier and not from the marker. It is known that meningiomas present a so high uptake of DOTATOC that 60 minutes from the intravenous administration are enough to obtain a clear meningiomas to background identification [42]. Regrettably, gliomas show a much weaker uptake to DOTATOC than meningiomas. The tracer uptake is so poor that PRRT could not be used for gliomas, since the dose to be injected, for an effective treatments, would cause a too high healthy tissue irradiation [43]. Anyway in case of β -RGS the tracer is not administered with the purpose to kill cancer cells, but to find them. A lower dose could be enough for the purpose, in particular considering the locality of the radiation that allows a clearest tumour masses identification. To precisely evaluate the uptake of DOTATOC by meningiomas, gliomas and the surrounding healthy tissue, finalized to an application of β -RGS, a statistic study was then performed on 23 patients from Istituto Europeo di Oncologia (IEO), Milan, 11 affected by meningioma and 12 affected by glioma [44]. In tab. 2.5 are reported the characteristics of all the patients affected by meningiomas. When the identification of multiple lesions was possible, each one was considered alone. All the patients, except the first two, were previously treated either by surgery or radiotherapy. In case of glioma, all the 12 patients were previously treated either with surgery, radiotherapy or chemotherapy. Their characteristics are showed in tab Standing the lower uptake of tracer, that causes a lower contrast on the images, in this case it was not possible to disconnect different lesions. To evaluate the uptake of the tracer from PET scans, Regions Of Interest (ROIs) were drawn on DICOM images. DICOM (Digital Imaging and COmmunications in Medicine) is a communication standard that defines how to codify a digital image in medicine. The images gave a two dimensional view of the tumour, so different slices were analysed to cover the entire tumour volume. Each slice in the DICOM images was 3.27 mm thick.

34 28 2. Medical applications Patient N of lesions W (kg) A (MBq) Diagnosis M atypical (gr II) M spheno-petro-clical M recurrent atypical M recurrent M recurrent M malignant M secondary M cervical M recurrent M atypical extracranial M atypical (gr II) Table 2.5. Characteristics of the sample of meningioma patients, showing for each one its number of lesions, weight (W), the injected activity (A) and the diagnosis. Patient W (kg) A (MBq) Diagnosis GB glioblastoma GB glioblastoma multiforme GB glioblastoma GB glioblastoma GB glioblastoma GB glioblastoma GB glioblastoma GB glioblastoma GB glioblastoma multiforme GB olygodendroglioma GB high grade glioma GB glioma Table 2.6. Characteristics of the sample of glioma patients, showing for each one its weight (W), the injected activity (A) and the diagnosis. For meningiomas, in case of patients with multiple lesions, each one was considered separately, and the ROIs were drawn to follow their border, taking care not to include healthy tissue nearby. For gliomas, between areas with high uptake there was often a necrotic area, due to previous treatments. In this case, standing the lower resolution of the image, following a conservative approach, this area was included in the ROIs. An example of ROIs for both cases is shown in fig The analysis of the images was made with the software AMIDE [45]. For each of the N s slices, the software gave the mean vales µ, the standard deviation σ, and the number of effective voxel contained in the ROI, N v. The specific activity of DOTATOC, µ, and the corresponding uncertainty σ µ for the whole lesion was computed as a weighted average:

35 2.3 Clinical cases of interest 29 Figure 2.7. Example of ROI for meningioma (left), high grade glioma (center), and healthy tissue (right) where w i = N v i σ 2 i µ = Σ iw i µ i Σ i w i ; σ µ = and i is a index ranging from 1 to N s. 1 Σi w i (2.1) Activity does not take in account distinction in weight and administered dose and the difference in times among the patients between the injection and the exam. These parameters are to be considered to compare the uptake among different patients. To this aim, the Standardized Uptake Value (SUV) is commonly used. SUV represents the ratio between the activity of the selected region and the radioactivity concentration in the hypothetical case of complete uniformly diffusion in the whole body. It is obtained normalizing the measured µ to the injected activity per mass unit, once corrected for the physical decay from the injection time: SUV = µ A 0 e 0.693( T P ET /T Ga ) P (2.2) where A 0 is the activity injected to the patient, T P ET the time interval between the administration of the radiopharmaceutical and the exam, T Ga is the half life of 68 Ga (68 min) and P is the weight of the patient. In case of RGS, the ratio uptake between the lesion and the surrounding tissue is a key parameter. The uptake of healthy tissue was estimated with the same approach used for malignant ones, analysing several ROIs on several slices for each patients. Considering the locality of β radiation, the ROIs were chosen, by means of the information from the CT, close to the tumour margins. Then a weighted average was used to evaluate the corresponding SUV NT. The Tumour non Tumour Ratio (TNR) was thus estimated as the ratio between SUV T and SUV NT. The results in case of meningiomas are shown in tab All the meningiomas showed good uptake of DOTATOC. About 70% of the tumours had a SUV greater

36 30 2. Medical applications than 2 g/ml. This figure means that a dose injection of 3 MBq/kg (activity normally used for PET) would produce a specific activity greater than 6 kbq/ml. Differences were spotted between several slices. Probably due to previous treatments there was not an uniform tracer distribution within the tumour masses. Uptake dissimilarity were found also between different lesions of the same patient, and this is reflected in the TNR, as shown in fig. 2.8, where each color-marker combination represents a different patient. The TNR for meningiomas resulted always greater than 10. Patient Lesion s number SUV T SUV NT TNR (g/ml) (g/ml) M M M M M M M M M M M M M M M M M M M M Table 2.7. SUV for both tumour and background in case of meningioma. Each lesion is reported alone. The TNR is also reported As expected, the uptake in gliomas was found weaker than in meningiomas. About 60% of the lesions showed a SUV of about 0.2 g/ml, that means that the injection of an activity of 3 MBq/kg would produce a specific activity of about 0.6 kbq/ml as shown in tab Also TNR values resulted smaller than the ones for meningiomas. With only one exception, TNR values were always greater than 4 and greater than 8 in one third of

37 2.3 Clinical cases of interest 31 Figure 2.8. TNR for each of the lesion analysed in case of meningiomas. Points with the same color and marker belong from the same patient the cases (fig. 2.9). It has to be noticed that these estimations are quite conservative, since clearer areas were included in the ROIs. This could lead to an underestimation of the the uptake of the whole lesion, and as consequence the TNR. Patient SUV T SUV NT TNR (g/ml) (g/ml) GB GB GB GB GB GB GB GB GB GB GB GB Table 2.8. SUV for both tumour and background in case of glioma and the corresponding TNR This study confirmed the uptake of the tracer in case of brain tumours. The possibility of the application of β -RGS with this uptake will be discussed in cap Neuroendocrine tumours Another natural field of application for β -RGS is the one relative to NETs, where PRRT with DOTATOC is used, as discussed in par

38 32 2. Medical applications Figure 2.9. TNR trend in case of gliomas. In this case each point represents a different patient NETs are neoplasms that arise from cells of the endocrine and nervous system. The function of neuroendocrine cells is to release message molecules (hormones) in the blood when receive a neuronal input. Hormones regulate body s physiology and behaviour. NETs inherit this ability of produce hormones, so can be responsible of serious illnesses. This type of NETs is called functioning tumours. Those that do not produce extra hormones are called non functioning tumours. NETs can arise in different areas of the body, although they are mostly located in the intestine, pancreas and lungs. The important thing is that, despite their different localizations throughout the body, due to their common origin they produce the same molecules and show similar receptors. Giving their variety their are grouped as follow: - NETs developed in the gut or pancreas are called gastroenteropancreatic neuroendocrine tumours or GEP NETs - Carcinoid, rare tumours with a slow growth, arise from digestive system and lugs - NETs developed in the pancreas are also called endocrine tumours of the pancreas. These include Insulinomas, Gastrinomas, Glucagonomas, VIPomas (producing vasoactive intestinal peptide) and Somatostatinoma Also rare types of NETs develop in thyroid gland, parathyroid gland, skin. NETs are rare, having an annual incidence of approximately per people, of which two thirds are carcinoid tumours and one third other NETs.

39 2.3 Clinical cases of interest 33 NETs usually develop slowly over a number of years. Commonly a NET, due to the lack of symptoms, is diagnosed when has already spread to another part of the body. Standing the late stage of discovery, with metastatic spread, often curative surgery is no longer an option, as well as external radiotherapy, considering the number of disseminated lesions Uptake evaluation: NETs DOTATOC is normally used for treatments of NETs with PRRT [46], so studies about the uptake are present in literature. Indeed the information required for PRRT are quite different from the ones for RGS. PRRT requires a preliminary evaluation of receptors by a PET scan with 68Ga- DOTATOC. After this first evaluation, the most important parameter becomes the uptake of other organs, firstly the kidney. In fact, in PRRT the activity to be injected to the patient is calculated on the basis of the dose absorbed by the kidney. It is the renal toxicity the adverse side effect to keep under control in the view of a balance between costs and benefits for the outcome of the patient. To obtain a precise evaluation of the tumour uptake to estimate the potential of RGS, a statistical study on DICOM images of patients affected by NETs at Arcispedale Santa Maria Nuova, in Reggio Emilia was carried out. The patient cohort was composed by 15 patients affected by primary or secondary NETs in different localizations, mainly within the abdomen. For 13 patients, 2 lesions were considered, as shown in tab The patients were under PRRT since, due to the number or recurrent nature of the lesions, they could not be treated surgically. After therapeutic administration of 177Lu-DOTATOC, a series of SPECT/CT scans of the abdomen, at about 0.5, 4, 20, 40, and 70 h after injection were taken. These scans are normally acquired to evaluate the dose absorbed by the organs and consequently adjust the further treatments. Following an approach like the one for brain tumours, DICOM images were used to evaluate the uptake of both normal organs and tumours at each time interval, with the aim to build a profile of this uptake as a function of the time from the injection. The analysis was performed using the SYNGO software. In this case, there was the possibility to create Volumes Of Interest (VOIs) instead of different regions on different slices. The 3-dimensional isocontour tool was used. Whit this tool, regions containing only voxel which value were at least 50% of the maximum value were created. A voxel is the 3D equivalent of a pixel, and in this study, the voxel volume was 0.11 ml (4.8 x 4.8 x 4.8 mm 3 ).

40 34 2. Medical applications Patient Weight Administered Lesion A Lesion B (kg) activity (GBq) Liver Liver Liver Liver Liver Bone Kidney Kidney Liver Liver Liver Liver Liver Pancreas Liver Kidney Liver Liver Bone Bone Bone Liver Liver Lung Pancreas Colon Liver Bone Table 2.9. Weight, dose administered and lesions studied for each of the 15 patients of the study All VOI had a similar volume of a few millilitres. Examples of VOIs in tumours and healthy organs are shown in fig In order to be sure to avoid necrotic areas, the VOIs were created on the zones showing higher uptakes in the lesions. For the other organs, healthy zones were selected, even in cases in which metastasis were therein present. The primary interest was the behaviour of the tumour respect to healthy organs, not respect to metastasis. The uptake of kidney, spleen, liver and a zone behind the spinal column (surely healthy muscle) was analysed. For each VOI, the software computed the total volume (V), the mean value of SPECT counts (µ) with its standard deviation (σ). SPECT counts needed to be converted to specific activity (A raw ) and this was estimated as: µ A raw = (2.3) kt scan V where k was a calibration factor equivalent to 11 cps/mbq needed to take in account detector non linearity for high activities (when saturation occurred), and T scan was the duration of the scan that lasted 1920 s. It has to be reminded that this study was finalized to application in RGS. For this reason A raw was converted in the activity of 90 Y expected at the time of the surgical operation. Since the study was made with 177 Lu, an the isotope planned to be used for RGS is 90 Y, corrections due the different half lives were considered

41 2.3 Clinical cases of interest 35 Figure Example of ROIs built on a SPECT/CT image using the isocontour tool in the case of a NET in the liver. It is possible to identify the VOIs of the two lesions (red and blue lines), the health liver (violet line), the spleen (green line) and the healthy tissue (yellow line) (T Lu =162 h; T Y = 64 h). A = A raw e 0.694[ T surg T scan ] T Y T Lu. (2.4) T scan was a factor that takes into account the time elapsed between the injection and the surgical operation, and was fixed at 24 h for reasons that will be shown later. Then, activity was converted in SUV: SUV = A ρa M 0 e T surg T Y (2.5) Where A M 0 was the administered activity per unit weight of the patient and ρ= 1 g/cm 3 was a reference density introduced to make the SUV nondimensional. The first goal of this study was to follow the uptake by tumoural and healthy tissues as a function of the time, to optimize the time required between injection and surgery. Since SUV already takes into account the correction due to the radioisotope half life, it is a natural parameter to analyse the time evolution. As shown in fig for a sample patient, a saturation effect after 24 h was spotted in the lesions. Different behaviour was found in the healthy tissue where sometimes, in combination with the biologic washout, an initial accumulation of the tracer was identified. This was in agreement with the studies available (fig from [47]), and dependent on the various characteristics of the patient like other pathologies and ongoing treatments.

42 36 2. Medical applications Figure patient 1 SUV as function of time after injection in lesions and healthy organs of Figure Examples of observed pharmacokinetic behaviours for DOTATOC: accumulation (blue line) and clearance (red and violet lines, according to the rapidity of the decrease)

43 2.3 Clinical cases of interest 37 To study the expected applicability of β -RGS to NETs, we restricted our patient sample to only those with NETs in the liver, for a total of 16 lesions from 11 patients of the cohort. According to the results on the time dependence of uptake for each patient, the DICOM image nearest the 24h interval from radiopharmaceutical administration was considered for this study. For RGS applicability, the specific activity of the tumour needed to be compared versus the background from nearby healthy tissues. To this aim, the profile of the counts along a line crossing each lesion was extracted with ImageJ (version 1.47) software. From these profiles, the maximum uptake of the lesion (µ MAX ) and the uptake of a region close to the lesion but clearly separated from it (the near background or µ 0 ) were estimated (respectively intersection between the profile and the purple and green line showed in fig. 2.13). The SPECT counts where converted into SUV with the same procedure aforementioned and then the TNR was estimated. The results are shown in tab Figure Example of ImageJ profile used to determine tumour uptake and near background uptake needed to evaluate expected performance of RGS technique in NETs. The 2 vertical lines correspond to µ MAX (violet) and µ 0 (green) The SUVs resulted very high. All patients but one showed a SUV greater than 10, and even with an administration of only 1 MBq/kg (a third of the standard PET dose) the expected value resulted greater than in case of meningiomas. Also the TNR resulted very promising, since all of them were higher than 6. Summarizing, in this chapter studies finalized to an effective use of β -RGS were presented. The necessity of a radiotracer that efficiently and selectively delivers a β emitters to the tumour has been solved with the choice of 90Y-DOTATOC. The uptake of this tracer in brain tumours (meningiomas and gliomas) and in abdomen tumours was studied. Results showed a promising uptake.

44 38 2. Medical applications Patient Lesion Lesion 24-h SUV 24-h SUV 24-h TNR 24-h TNR A B lesion A lesion B lesion A lesion B 1 Liver Liver Liver Liver Liver Bone N/A 4 Kidney Kidney N/A N/A 5 Liver Liver Liver Liver Liver Pancreas N/A 8 Liver Kidney N/A 9 Liver Liver Bone Bone N/A N/A 11 Bone Liver N/A Liver Lung N/A 13 Pancreas 85.9 N/A N/A 14 Colon Liver N/A Bone 9.4 N/A N/A Table SUV after 24 h for the liver lesions and the corresponding TNR. N/A, not applicable, when the lesion was not in the liver

45 Chapter 3 Scintillator s characterization Different aspects must be taken into account when designing a detector for β RGS. Essentially, the device must have high efficiency on electrons down to low energies, to allow low dose injection. It must be as transparent as possible to photons, to avoid the Bremsstrahlung radiation. In addition, the entire detecting system should be arranged in a thin device to result into an handy tool for the surgeon. A compact counter, based on a scintillator, is expected to fit these requirements. The choice of the active material plays an important part in fulfilling the apparatus requirements. Scintillators are divided in two families: organic and inorganic. Usually, inorganic crystals have a high light production, high Z, and slow scintillation ( 100 ns). Organic crystals have lower densities and shorter decay times. Moreover, their low Z make them usually poorly sensitive to photons. 3.1 Doped para-terphenyl The organic crystal para-terphenyl doped with 0.1% diphenylbutadiene (DpT) was selected as the most appropriate candidate for the creation of the probe [48]. Paraterphenyl (1,4-diphenylbenzene) is an aromatic hydrocarbon isomer, formed by three benzene rings (fig. 3.2). Pure terphenyl is a white crystalline solid insoluble in water. Undoped para-terphenyl crystals are of limited use because of their low light output. Introduction of special dopant ensures the light output to be increased by 4-5 times [49]. DpT crystals retain all basic advantages of organic scintillators with a characteristic wavelength peaked at 450 nm (fig. 3.2). Table 3.1 shows comparative data for DpT and other similar organic crystals. From the table is evident the large light output possessed by DpT. Due to its short light attenuation length, DpT is normally used as dopant for plastic scintillators instead of pure crystal. Aiming at conceiving a small electron detection probe, with an active volume of the order of few millimetres, a short λ does not represent a problem when counterbalanced by a convenient gain in light production. 39

46 40 3. Scintillator s characterization Figure 3.1. Para-Therphenyl structure (C 18 H 14 ) Figure 3.2. Spectrum of light transmitted through the DpT sample (bottom), and in absence of the sample (top). The light source was a Deuterium UV Lamp. The DpT emitted light is in the range nm, a zone commonly well covered by standard PMT

47 3.2 Stability to temperature variation 41 DpT in two different status, polycrystalline and monocrystalline (purchased by DETEC-Europe) was analysed. The polycrystalline DpT consisted in a material obtained from DpT powder under pressure, whereas the monocrystalline DpT was a pure crystal. In fig. 3.3 disks of both the compounds are shown. The density of the polycrystalline disk was 1.16 g/cm 3 while the density of monocrystalline disk was 1.14 g/cm 3. Figure 3.3. Left: pure monocrystalline DpT disk; right: polycrystalline DpT disk, for which it is evident the granular structure of the pressed powder. Structural differences modify the optical proprieties Antracene Doped p-terphenil Stilbene Density [g/cm 3 ] Light output [10 4 photons/mev] Decay time [ns] Table 3.1. Comparison between the main characteristics of typical organic scintillators, from [50] 3.2 Stability to temperature variation Tests were performed to quantify the eventual temperature effect on DpT although the portable detector was designed to be used in surgical room, where temperature for its importance (i.e. occupant comfort, infection control), is monitored to be compliant with specific guidelines ( 20 C [51]). The temperature stability of the material was tested with a Peltier cooling module, in the range of temperature from 10 to 30 C. Aiming to estimate the response of the device in absence of light, a DpT rod (diameter 2 mm, height 4 mm) was coupled to a PMT (Photon Multiplier Tube, modules H10720 [52] - later on, when not otherwise specified, the text refers to this model) with an optical fibre.

48 42 3. Scintillator s characterization The PMT s dark count rate is function of the absolute temperature, so it was tested separately from the crystal. In each measures, only one of the two components was inserted into the cooling module, while the other one was exposed to ambient temperature. As shown in fig. 3.4 while the scintillator results insensitive to temperature variations, the PMT shows a dependence compatible with the one reported by the manufacture in the data sheet [53]. To manage this effect, the PMT was located inside a thermal insulated container. Figure 3.4. Left/Right: Test on stability of the dark counts rate to temperature variations in the range C for the scintillator/pmt separately. Configurations with different electronics thresholds were investigated 3.3 Light attenuation length Considering a photons beam impinging on a material, the starting intensity I 0 and the intensity after a travel x inside the material I x are related by the following equation: I x = I 0 e x/λ (3.1) A small Light Attenuation Length (λ) means that the light is quickly attenuated as it passes through the medium, while a large attenuation coefficient means that the medium is relatively transparent to the beam. This parameter has an important influence on the detector dimensions. The DpT light attenuation length was measured using disks with a same diameter, 25 mm, but different thickness -2,3,4,6,8,10 mm- illuminated by a LED (Light Emitting Diode). To set an uniform illumination of the disk, the pulsed light emitted by a blue LED [54] (peaking at 473 nm) was diffused by a thin Teflon disk that guided the light into a black cone to a glass diffuser [55]. The led was driven by an external wave form generator (square waves, frequency 3 khz, 30 ns width, V height). The outgoing light was collected with an optical fibre (diameter of 1 mm) coupled to a SiPM (sensl B-series 10035, with spectral sensitivity peaked at 500 nm [56] -below the text refers to this model, unless otherwise specified). The fibre was mounted on a

49 3.3 Light attenuation length 43 motorized system spanning a 10x10 mm 2 surface in 1 mm step. The set-up is shown in fig The SiPM bias voltage and signal shaping were provided by a custom board, and the resulting pulse was acquired with an oscilloscope (Teledyne LeCroy waverunner 610Zi [57]). All the measures were collected inside a lightproof black box. Figure 3.5. Internal view of the black-box used. Starting from the left, the diffusive black cone with LED inside, the optical fibre supported by a two axis motorized system for the scan, and SiPM inside an aluminium body to handle and protect him. In the insert the same set-up is shown by a different perspective to display the position of the DpT (the white disk) Before the data taking, the uniformity of the light emission after the glass diffuser had to be verified. To this scope, the pulse charge distribution was collected in each point of the map. The acquisition was triggered by the pulse driving the LED. Due to the intrinsic resolution and linearity of the read-out chain (SiPM plus the pre-amplifier board) the pulse height of any event was proportional to the number of the outgoing photons entering the fibre. The distribution of the integrals of these pulses was acquired and analysed using TSpectrum, a class of ROOT, a data analysis framework [58]. In this way it was possible to identify each peak of the distribution and compute an integral for each Gaussian (fig. 3.6). A linear calibration allowed to found a correspondence between the position of the peak (mean value of each Gaussian) and the number of cells activated for pulse, a value equivalent to the number of photons reaching the SiPM standing the low intensity of the LED. Combining these two values the Poisson distribution of the number of photons was obtained (fig. 3.7), where the height of each bin was the integral of the corresponding Gaussian. The map in fig. 3.8 shows the distribution of the light emission. The measure was repeated inserting DpT disks of different thickness between the glass and the optical fibre. In this analysis, edge effects were neglected considering that the disk area (diameter 25 mm) was much wider than the 10x10 mm 2 area investigated in the scan. For these scans both the disks of poly and monocrystalline DpT were used. The intensity of the light coming out of the diffuser was proved to be constant at

50 44 3. Scintillator s characterization Figure 3.6. Distribution of the pulse charge generated by the SiPM excited by a LED pulse of fixed amplitude. Depending on the number of photons reaching the SiPM active surface, zero, one or more cells contribute to the output signal. The measured spectrum reflect this quantization with the first peak corresponding to "zero" cells, the second to the activation of one cell and so on. Figure 3.7. The distribution of the number of photons for pulse acquired by the SiPM follow a Poisson law, as the fit shows

51 3.3 Light attenuation length 45 Figure 3.8. Distribution of the light intensity after the glass diffuser, percentage change respect to the mean value different distances between the fibre and the optical glass (0, 4, 10 mm), so no geometrical correction factors were necessary to compare the results obtained using different disks. The number of photon resulted homogeneous among the disk surface, as it is shown in the fig In this case - 2 mm thick monocrystalline DpT- in less than the 4% of positions a non-uniform higher than 10% was acquired. For each disk, the mean value of the photons number computed on the entire map was calculated. The trend is reported in fig To estimate the value of λ, the dependence of I x normalized to I 0, whit x the material thickness, was fitted with an exponential. Two different values of λ were found, 5.03 ± 0.23 mm for the polycrystalline and ± 0.88 mm for the monocrystalline DpT. Figure 3.9. Distribution of the light intensity after 2 mm of monocrystalline DpT as percentage change respect to the mean value In case of the monocrystallin DpT, the 8 mm disk was excluded from the fit, being

52 46 3. Scintillator s characterization Figure Left/Right the decrease of the light intensity increasing the thickness of material to cross, for the mono/poli crystalline disks the rate acquired too low. An error in the alignment between fibre and disk was investigated as cause of this value out of the trend. Misalignment changes the field of view of the fibre and its angular acceptance, resulting in a lower number of photons acquired. To confirm this hypothesis, a second run of measurements was taken acquiring only a value in the middle point of each disk, given the uniformity of the surface. Avoiding the scan, the correct alignment between fibre and disk was assured. In this configuration all the disks showed a comparable behaviour an a value of 17.9 ± 1.0 mm for λ was found (fig. 3.11). Figure λ s estimation for mono-crystalline DpT, considering only one point for disk The attenuation measured so far includes both the effect of absorption and diffusion of light crossing the media. This multi-effect parameter is a critical point for the optimization of the design of a counting device. In case of an imaging application, diffusion alone is expected to be the most relevant parameter. To uncouple this parameter, expected to be quite different for mono and poly crystalline DpT, further measures were acquired.

53 3.3 Light attenuation length 47 Using the same set-up, a tiny copper mask (300 µm) was inserted between the glass and the DpT disk. The mask was drilled with holes of 1 mm diameter spaced at different distances between them (1-2-3 mm, as shown in fig. 3.12). Figure The copper mask used to evaluate the effect of the diffusion The resulting scans of the surface, with a step of 0.5 mm, are shown in fig The plot on the left refers to the scan over a 2 mm thick monocrystalline DtP, the one on the right shows the same scan over a 2 mm thick polycrystalline disk. The reconstruction of the position of the holes resulted easier with the monocrystalline DpT and this material was considered to fit better for an imaging probe. In order to estimate the resolution, the Y slice at y 3.5 mm (fig. 3.14) was fitted as a convolution of 3 gaussians: A 0 gaus(µ 0, σ 0 ) + A 1 gaus(µ 1, σ 1 ) + A 2 gaus(µ 2, σ 2 ). (3.2) The central spot was surrounded by the other spots and collected light from a wider area. The resolution estimated from the central Gaussian was 0.72 ± 0.08 mm. The two external spots did not receive contributions from all the sides. The sigma value of corresponding Gaussians represents the expected resolution at the edges of the active spots, close to the border with lower uptake regions. Diffusion in the polycrystalline DpT resulted too high and was not possible to identify the spots. On the contrary, it was possible to acquire a clear image of the mask in case of the monocrystalline disk. Figure Left/Right the reconstructed images in case of mono/polycrystalline disk with a LED as percentage change respect to the maximum value

54 48 3. Scintillator s characterization Figure Y projection of 2 mm monocrystalline DpT maps for Y 3.5 mm. The resolution over the three spots (1 mm diameter) was estimated as the sigma of the Gaussian 3.4 Light collection Light collection impacts the probe performance influencing the minimum detectable electron energy and the efficiency on the detection of events over threshold. The use of different materials to wrap the DpT was studied, to increase as much as possible the number of photons arriving at the sensor when a particle crosses the detector. The wrapping of the active material with reflective or diffusive foil is a standard method. The effectiveness of the two approaches depends on the geometry and optical characteristic of the crystal. The light collection was measured using disks of a same diameter, 25 mm, but different heights -2,3,4 mm-, a 90 Sr diffused source and a SiPM. The starting points were the respective counts for each naked disk. The set up consisted of a disk on the top of a source of 90 Sr directly coupled with a SiPM (active area 1x1 mm 2 ) located in the center of the disk. In this configuration the polycrystalline disks respect to the monocrystalline with the same thickness, allowed to acquire a higher value of counts, as shown in tab Thickness [mm] Poly- crystalline Rate [Hz] Mono- crystalline Rate [Hz] Table 3.2. Rates of different thickness disks with the same set-up (source, optical coupling, electronics gain and threshold) To try to reduce this difference (factor 2 between the 2 mm disks), wraps of different reflective materials for the mono DpT disk were tested (tab. 3.3) : Aluminium (10 µm thickness); Tyvek (20 µm thickness); Polytetrafluoroethylene (PTFE or Teflon

55 3.5 Crystal height optimization µm thickness); Mylar (10 µm or 20 µm thickness). As the table 3.3 shows, the double Mylar wrapping proved to be the best configuration to reduce the gap, with a recover in the rate equivalent to 32%. In case of mono DpT 4 mm disk, the rate with a double Mylar wrapping increased from 40 to 70 Hz. On the contrary, the rate increase of poly DpT disk resulted less affected by the surface reflectivity, with values increasing from 330 to 400 Hz (17%). Nothing Aluminium Tyvek PTFE Mylar Mylar x2 Rate [Hz] Table 3.3. Effect on rate on a disk of 2 mm mono DpT of different reflective materials As alternative to the wrapping, that in any case adds some material to the probe electron entrance window, a simple polishing of the crystal surface has been tested. This approach was found not competitive with the wrapping giving at most a 3% increase in the rate with respect to the raw surface. Regardless of its short λ, poly DpT allows higher rates, thus resulting more appropriate for counting devices 3.5 Crystal height optimization The size of the sensitive part of the detector is established by the light attenuation length but to maximize the sensitivity to β particles, the optimal thickness of the scintillator was determined experimentally. A cylinder of DpT (polycrystalline) with diameter of 2.1 mm was connected to the PMT by one optical fibre and the thickness of this scintillator was gradually reduced, with a non reversible process, starting from 4 mm until the counting rate on a point-like 90 Sr source with activity of 370 Bq was maximized. The thickness was reduced using wet sandpapers with different grids (DpT was not hygroscopic), and the smoothing of the surface was checked after each cut with a digital microscope. The set-up was located inside a black box to ensure lightproof. Fig shows the counting rate acquired on the Sr-source as a function of the para-terphenyl thickness. In these experimental conditions, the optimal point was found to be around 2 mm. Actually, it has to be noticed that this value is the optimal working point for 90 Sr while, for clinical application, the use of 90 Y was planned. 90 Sr is a long-lived β emitter (half-life 29 years) with maximum decay energy of 546 kev and no gamma radiation. It exists in secular equilibrium with its daughter isotope 90 Y resulting in the emission of two β particles. Therefore the resulting energy spectrum is more populated at low energy.

56 50 3. Scintillator s characterization Figure Probe counting rate as function of the detector thickness 3.6 Sensitivity to photons The DpT photon sensitivity is an important parameter to evaluate the effect of Bremsstrahlung radiation in clinical practise. This parameter also affects the possibility to use β emitter with a small percentage of γ decay. The sensitivity of DpT to gammas of energies ranging from 600 KeV to 1100 KeV, was tested on a DpT disk with 10 diameter and 3.1 mm thick, directly coupled with a SiPM placed at the center. Three point-like sources: 133 Ba emitting photons with energy ranging from 80 to 350 kev, 137 Cs with gamma emission at E γ =662 kev and 60 Co with E γ1 =1170 kev and E γ2 =1330 kev were used. To avoid signal from electrons, in the case of Cs decays, three copper layers with 350 µm thickness were inserted in sequence between the source and the disk, and the measurements were repeated at each step. The efficiency for the three sources is shown in fig Except for the first measure on the Cs source, introducing the copper absorbers implies a very small decrease in rate compatible with the photons attenuation in copper and the change in geometrical acceptance. The efficiency was evaluated by comparing the measured rate with the nominal activity of the sources rescaled for their lifetime at the time of the test, namely 36 kbq for 133 Ba, 181 kbq for 137 Cs and 3.2 kbq for 60 Co. In case of Cs, without copper shield, the total rate for photons plus electrons was 1761 Hz. To separate the two populations, the estimated number of photons from the linear fit was extracted. In this way it was possible to compute the number of counts due to electrons and estimate the efficiency considering only the most energetic fraction of electrons (1.174 MeV as shown in the decay scheme in fig. 3.17). It was estimated that 0.51 MeV electron had not enough energy to reach the detector since the disk was wrapped inside an aluminium foil (100 µm) to ensure lightproof. As a further check of the results, the SiPM resulted insensitive to a direct exposure to the photons. The sensitivity to the photons emitted by the 133 Ba source was below 0.1%, whereas

57 3.6 Sensitivity to photons 51 for the more energetic γs from the 137 Cs and 60 Co sources the sensitivity was still below 1%. In case of Bremsstrahlung radiation the photon energy spectrum is expected to peak at 100 kev. This measure showed that the DpT sensitivity to this type of radiation decreases according to the energy of the photon. The sensitivity value of 0.1% can be considered a reasonable upper limit in case of Yttrium Bremsstrahlung. In conclusion an intraoperative β probe will not be sensitive to the Bremsstrahlung photons and therefore the effectiveness of the RGS technique would not be affected by this background. Figure Probe sensitivity to photons emitted by three different sources of photons ranging from 80 to 1330 kev. The measurements were repeated after insertions of copper layers with thickness of 350 µm between the source and the disk to absorb the electron component of the Caesium emissions Figure Cs decay scheme

58

59 Chapter 4 Probe design The potentiality of the DpT as active element of a β probe and the technical constraints set by the clinical applications were studied making specific prototypes exploiting different solutions on the light detection technique, the read-out electronics and the feedback the system is capable to provide. In this chapter the basic performances of the prototypes on point like and wide area β sources are reported. 4.1 Guidelines As already mentioned the system was thought for oncology. This sets a number of guidelines the development of any prototype must follow: - result in a handy tool (thin and light) - provide a real time feedback of the detected activity - be capable to detect tumour remnants of the order of 0.1 ml when the injected dose is less or at most equal to that used in imaging application (PET, SPECT) - have a read out electronics decoupled from the probe body with, possibly, a wireless monitoring device. 4.2 Probe1 The first system developed (named Probe1) aimed at a complete decoupling between the powered part of the device (photon detector and read-out) and the one in contact with the patient (scintillating crystal). The solution adopted is a rod of DpT (for detail on the material see chap. 3) coupled with an optical fibre guiding the scintillation light to a miniaturized Photo Multiplier. The core of the first prototype was a small cylinder of DpT with a diameter of 2.15 mm and 4 mm thick done pressing some DpT powder inside an hole drilled into a larger, white, PVC cylinder. This method to create a small active volume was the first technique adopted before demonstrating that was possible to cut and shape small crystals by machining a bulk with the appropriate tools. 53

60 54 4. Probe design Figure 4.1. Wrapping of the mylar at the top of the active material Figure 4.2. The massive lateral PVC protection used An optical fibre (1 mm of diameter, 60 cm long) was glued with cyanoacrylate at the center of one face of the scintillating material. On the other face a thin reflective mylar foil (10 µm, see fig. 4.1) was fixed with optical grease and black tape. To increase the lateral shield and give robustness to the assembly the compound was encapsulated in a 21 mm diameter black PVC cylinder 13.3 mm height, as shown in fig Light tightening was obtained by covering the tip surface with 200 µm black tape on one side and coupling the back side with 10 cm long plastic tube (thermoforming material) to simulate a pen-like probe to facilitate the handling of the device during the test. The light was collected by the optical fibre and carried to a PMT [52] powered by a bench DC power supply. The PMT output was sent to a digital oscilloscope to characterize the PMT response (dark counts versus bias voltage), set the optimum gain and the trigger threshold. The scope of this first assembly was to prove that a scintillator made by a DpT works, check the effectiveness of the fibre to DpT coupling and give some experimental data to start with the development of a Montecarlo simulation (see sec ) of this class of devices. The Probe1 response has been measured sliding a point-like sealed 90 Sr source under the tip. The source, moved in step of 1 mm by a step motor was in contact with the probe head and the rates were measured with a digital oscilloscope. The simple set-up is shown in fig This scan, as expected, shows a bell profile and, with a Gaussian fit, a peak value of 30 Hz and a sigma of 2.6 mm were found. The device, although non completely lightproof (the weakest point was the thermoforming material cover) and not very handy, proved the possibility to use DpT as scintillator and resulted a good starting point for further developments. A restyle, finalized to solve these weaknesses, was made. The active volume (reduced

61 4.2 Probe1 55 Figure 4.3. The set-up created to measure the Probe1 response. The source (the yellow disk) was fixed on an stepper motor and was moved under the probe head to a 2.1 mm diameter, 1.73 mm height rod as discussed in sec. 3.5) was mounted on the top of an aluminium body to allow an easy handle of the device. To ensure the lightproof and avoid mechanical stress to the fibre, the aluminium body was filled with a black glue. Always to increase the handiness of the tool, the lateral cover on the top was reduced. A tip of black PVC was used, and it provided a lateral shield of 0.6 mm and a frontal shield of 390 µm (fig. 4.4). Figure 4.4. The prototype of the intraoperative β probe. In the insert is shown the core of policrystalline DpT. The active material was encapsulated inside an easy-to-handle aluminium body as protection against mechanical stress and was protected against light by a thin PVC layer Great efforts were put also in the upgrade of the front-end electronics. A specific board based on Arduino Due was built [59]. The signal coming from the PMT was

62 56 4. Probe design integrated by a RC circuit (with a gain of 45 mv/pc and a τ of 1 µs) and sent to a comparator. The threshold was set by an external trimmer. The resulting square wave was then processed by the microprocessor. The software allowed to show the rates of an external tablet with a wi-fi connection. There was also the possibility to send the discriminated signal to standard Caen modules. In this way the use of oscilloscope as frequencymeter was avoided, increasing the stability and reproducibility of the measures. The front-end was stored in a portable box, improving the overall portability of the system, avoiding the necessity of a bench DC power supply. The handy and lightproof final prototype was compatible with a standard submillimetric sterile covering film for surgical environment First test with liquid Yttrium Purpose of the test was to verify the performance of the detector using a source of 90 Y, the tracer planned to be used in clinical application (see sec. 2.2), being the previous tests made on a 90 Sr source. 90 Y in saline solution, the same used in clinical applications, was available. 90 Y has an half life of 64 h so, respect to the case of 90 Sr (half life 28.8 y) a different set-up has to be made being sealed source not available. The range of activity concentrations from 22 to 5 kbq/ml was explored, to include the target value of 20 kbq/ml in patients affected by meningioma, as estimated by analyses of PET images after administration of 3 MBq/kg of 68 Ga-DOTATOC (corresponding to an administered dose of 210 MBq for a 70 kg patient). Holes with different dimensions were drilled in a plastic plate and filled with the radioactive saline solution. The dimensions of the holes are reported in tab The uncertainty of the dimensions of the holes was 0.01 mm. To avoid effect of surface tension a small quantity of tensioactive solvent was added, and the flatness of the water surface in each holes was checked with a digital microscope. The probe was fixed in vertical position over the plate at an heigh of 200 ± 10 µm (probe-plate distances of 50 and 150 µm were investigated too). A step motor, controlled via PC with a code written in LabView [60], rotated the disk under the probe. At the optimal working point, the background rate due to the PMT dark counts and casual events was measured to be 0.2 counts per second. It was checked to be stable when operating at room-temperature and with ambient light conditions. The 0.1 ml volume phantom, referred to as RESIDUAL, had dimensions compatible with residuals well identified with the PET. The smallest hole, referred to as OPBG, too small to represent a clinical case of interest, was aimed to identify the smallest volume detectable by the probe. Three cylindrical phantoms, with same diameter but different height, 1, 2, and 3 mm - referred to as H1, H2, H3 respectively - were created to evaluate the effect of the tumour residual depth on the probe response. Finally, the largest one, referred to as CALIB, was used as reference during the scan.

63 4.2 Probe1 57 Name H V Rate [Hz] Rate [Hz] [mm] [mm] [mm kbq/ml RESIDUAL OPBG H H H CALIB Table 4.1. For each of the six holes: name, dimensions, rates measured at the beginning and at the end of the measures. The rates at the nominal activity of 22 kbq/ml were acquired with a nominal distance probe surface of 200 µm and respectively the rate acquired at 5 kbq/ml with a distance of 50 µm A blind scan to simulate the surgeon exploring the area looking for residuals was performed to test the probe performance in a simplified "clinical configuration". In this configuration, the background was due only to device s dark counts, while in clinical cases the main source of noise should be related to the uptake of the healthy tissues surrounding the lesion. The motorized plate was rotated by a complete turn with 1 degree step angle (corresponding to 1.5 mm step along the circumference) recording rate measurement for 3s at each step. All the phantoms were clearly detected at the 16 kbq/ml 90 Y activity concentration as shown in fig The same scan was repeated reducing the time acquisitions to only one second, and also in this case all the spots were identified. The absence of a signal when the probe was located between the samples demonstrated the insensitivity to any long range radiation that could be produced by the activity of the phantoms. The effect of an off-axis measurement was verified moving the probe away from the phantom center. The test (reported in fig. 4.6) showed that the rate was reduced by a factor 2 respect to the peak when moving the center of the probe 0.5 mm far from the phantom edge, independently from the holes dimensions. This test proved the lateral shielding effectiveness of this prototype, a shielding able to make the probe insensitive to electrons coming from the sides so improving the tumour spotting capability and gave a first indication about the possibility to identify two near spots in clinical cases. To investigate the possibility to extract useful information from energy deposited, charge energy spectra were acquired with the probe centred over the three holes H1, H2, H3. The normalized charge spectra are shown in fig Differences were not appreciated among them, but it was not the same for the rates (tab. 4.1). The rates difference shows that the probe is sensitive to a depth deeper than 2 mm. In case of H3, the

64 58 4. Probe design Figure 4.5. Blind scan simulating surgeon exploring the area for hot spots. The counts were measured during a complete turn of the phantom disk (step of 1 degree; acquisition time of 1 s for each step; 90 Y activity concentration: 16 kbq/ml; distance between the probe and the phantoms: 50 µm) Figure 4.6. Rate profiles acquired moving the probe away from the centre of the phantoms with step of 1 mm and acquisition time of 200 s. The 90 Y activity concentration was 16 kbq/ml and the distance between the probe and the phantoms was 50 µm.

65 4.2 Probe1 59 electron population arriving at the probe was the same that in case of H2 plus the component of electrons from a further millimetre. Consequently the spectrum was expected to be enriched at low energies. It was impossible to notice this effect. This measure confirmed the uselessness to acquire energy spectra, being the dimension of the device too small to contain the energy release. It has to be reminded that Probe1 was designed as a counter, not as a calorimeter. Figure 4.7. Superimposition of the normalized charge spectra acquired over the three holes named H1, H2, H3 The rates trend was checked during the week of data taking. As shown in fig. 4.8 the rate decreased with the time in disagreement with the 90 Y decay law (2 t h/64 ) as confirmed by the bad value of χ 2. A disagreement was expected since water evaporation during the test occurred. Evaporation caused a volume decrease of the phantoms, changing in this way the distance between probe and source and reducing consequently the solid angle. Unlikely the liquid Yttrium at disposal was not enough for a complete refill of all the holes for all the week before each scan (only a small volume of 3 ml was bought to remain under the law limit about transportation of radioactive material). Anyway the trend allowed to affirm that in case of evaporation, Yttrium settled on the plate, and only the water vaporized. After this test, Probe1 underwent another upgrade to increase the efficiency. The frontal shield of PVC (390 µm) presented an unacceptable thickness, reducing the solid angle and cutting the low energy electrons, with a huge effect on the rates. A new cover with a 10 µm aluminium shield was realized, obtaining a good resistance and light tightness with negligible impact on the dark counts (the dark counts rate resulted to be < 0.3 Hz). On the point-like 90 Sr reference sources with nominal activity of 370 Bq, the rate increased by 21.2 %, from 37.8 ± 0.6 Hz to 59.0 ± 0.8 Hz.

66 60 4. Probe design Figure 4.8. Rates acquired (500 s/point) during a week scan, over one of the holes filled with the radioactive solution (RESIDUAL). As expected, the rate decreases following the 90 Y decay law 4.3 Probe4 A second system was developed (named Probe4) to evaluate the effect of a bigger active volume. A greater DpT rod was coupled with four optical fibres guiding the light to a PMT. The core of Probe4 was a polycrystalline DpT disk with a diameter of 5.1 mm and a height of 3 mm. The scintillator tip was shielded from radiation coming from the sides by a black PVC ring with external diameter of 11 mm (fig. 4.9). A 10 µm-thick aluminium sheet was used to cover the detector window to ensure light tightness. This assembly was mounted on the top of an easy-to-handle aluminium cylindrical body (diameter 8 mm and length 14 cm). The larger DpT surface - respect to Probe1 - allowed to glue four optical fibres (with a diameter of 1 mm), increasing the total light collection efficiency. A specific PVC black disk was made to tie the fibres (with a gain for the overall handiness) and join the end of the fibres with the PMT window, ensuring stability and lightproof of the coupling. Particular attention was paid to the cut and polish of the fibres optical surface each of one was checked with a digital microscope. The electronics chain used to acquire the waveforms was the same of Probe1. The final assembly of the probe with the tablet used for the read-out is shown in fig The first test with liquid Yttrium gave important indications about optimal active volume dimensions. While it is possible to fix the optimal height of the crystal on the basis of experimental data on energy release and light attenuation length, constraints on the diameter were subjective. The two most relevant parameters that drive the decision, handiness and field of view, are in contrast. To increase the handiness of the tool, a smallest as possible diameter is required, while to increase

67 4.3 Probe4 61 Figure 4.9. The active material of Probe4 wrapped into a black PVC and coupled with four optical fibres Figure Images of probe 4. The white cover was used to protect the fibres. It is shown the tablet used to acquire the counts the field of view, and consequently reduce the time needed to explore an extended area, lateral dimension has to be increased. In Probe1 the first requirement was strictly applied, and the prototype resulted unsuitable to scan a "wide" area, requiring too much time. In Probe4 a different compromise was tested After-pulse A set of measures was planned to define the Probe4 s proper operating conditions. The probe was fixed over the 90 Sr point source and the rates as a function of the threshold were acquired using a standard chain of Caen modules. The scan was made setting the PMT s gain values at V (the maximum value according with the data sheet was 1.1 V). The counts obtained are reported in fig with blue and red dot markers. Two different trends were identified, one for the low threshold region and one for the high threshold region. Considering the activity of the source, the value of the red dots overcame the possible expected value so these values were associated to an electronics bias. Checking the waveforms with the oscilloscope, in this gain-threshold region trains of smaller pulses after the trigger event were spotted in most of the cases. This effect was investigated also for Probe1, (the counts acquired are marked with black and green squares in fig. 4.11), but resulted less important, and this was associated to the lower light collection of the probe, that resulted in a less energetic trigger event. To evaluate the time distribution of after pulse events, a window of 50 µs was opened in the oscilloscope after the trigger event, and the mean number of after-pulses and their time distribution were recorded. In fig is reported the probability to

68 62 4. Probe design Figure The trends of the rates as a function of the threshold for Probe4 (dots) and Probe1 (squares). In each trend two different populations, at high and low threshold levels were found, and fitted with a specific exponential (marked with a different color code) acquire at least one after-pulse in the same time window as function of the threshold, while in fig the time distribution of the after-pulse respect to the trigger is reported. These plots show two possible ways to cut of this events: increase the threshold or insert a veto. As shown, a veto time of 4 µs was enough to reduce the fraction of after pulse to less than 1%. Indeed the electronics box s design did not take into account the possibility to use a veto, so this option was not undertaken. To eliminate this effect, the electronics threshold was fixed where the percentage of after-pulse was the 2%. With this procedure, the optimal working point of Probe4 was fixed. Figure Fraction of trigger events in which there was at least one other pulse in the same time window (log scale) Figure Time distribution of the pulses after the trigger (log scale)

69 4.3 Probe Field of view With the aim to investigate the Probe4 field of view, a bunch of tests were setted. These tests evaluated the effectiveness of the PVC lateral shield, the resolution of the probe as a function of the solid angle (both in water and air) and the detection depth. To verify the effectiveness of lateral shielding, the point-like source was set to illuminate the crystal trough the PVC shield. The alignment between DpT and the source was assured with a two axis scan. The maximum value of rate acquired in this scan resulted to be 7.2 ± 0.8 Hz. This value was compared with the reference rate acquired when the source was located in front of the probe, that resulted to be ± 1.0 Hz. The 3 mm thickness PVC shield proved enough to reduce the rate of a factor 20, preserving at the same time the handiness of the tool. The possibility to identify point residuals even when probe is not in contact with the tissue was evaluated studying the Probe4 resolution as a function of the solid angle. The probe was mounted on a motorized linear actuator ensuring position accuracy of 1.5 µm and horizontal scans were performed over the point-like 90 Sr source in air. Different distance probe-source were tested, ranging from 350 µm to 1.8 mm. The profile of the sources was reconstructed collecting measures in 10 s/position with steps of 1 mm. In fig the reconstructed profiles are reported. The values were fitted using a Gaussian distribution to extract the σ values that changed from 2.8 to 4.4 mm as a function of the distance. These scans showed how spatial resolution depends on the distance between the probe tip and the surface. Due to the bigger dimensions of the active volume, Probe4 had a resolution lower than Probe1, but still an area of resection compatible with the technical limits of surgical operations. Figure Probe4 s resolution over a point-source as a function of the distance between probe and source

70 64 4. Probe design To confirm the considerations about the applicability of Probe4 the same scans were reproduced in water. Standing the approximation between human body and water, these measures made possible to evaluate the discovery potential in real cases. As shown in fig 4.15, whereas in air the sigma increased with the distance due to geometry, in water this effect was mitigated by the absorption power of the medium. Independently from the depth of the source the σ was found to be lower to 3 mm. This is the maximum distance from which the probe is expected to identify a point size residual during the operation. Figure Values of the sigma of the Gaussian profiles obtained increasing the thickness of water between the probe tip and the point like 90 Sr source The previous discussed tests were focussed on the evaluation of the lateral field of view of the probe. To get a complete comprehension of how the probe operates in the surgical field, the detection depth has to be characterized. To this aim, scans over an 90 Sr extended-source (2.54 ± 0.15 kbq activity) in water and air were performed. The source active surface had a diameter of 16 mm, thus simplifying the alignment between the probe tip and the source. Fig.4.16 shows the counts decrease in air (yellow) and water (blue) increasing the thickness of the material between the probe and the source. The scan in water gave a rough estimation of the tumours depth detection in body (fig. 4.17), that resulted to be 8.3 mm. This value was defined as the intersection between an exponential fit on the experimental data and a line that represented the value of the dark counts. The scan was used to estimate the efficiency of the device using a FLUKA Monte Carlo simulation (about FLUKA see sec ). The agreement between data and simulation was first verified on the scan in air, then used to reproduce the counts endpoint in water. The simulation shows that the efficiency rises at energies above 540 kev (energy at which the curve reaches half of its maximum) while for higher energy reaches a plateaux with a value of 70%.

71 4.3 Probe4 65 Figure Measured rates as a function of the distance of the probe from the diffused 90 Sr source. The scan was performed in water (blue) and in air (yellow) Figure Zoom of the endpoint of the scan in water. An intersection between an exponential fit and the values of dark counts allowed to estimate the maximum detectable distance in water Second test with liquid Yttrium Attempting to obtain a direct comparison of the effect of a greater active volume, the same test with liquid phantoms made for Probe1 was planned for Probe4. In order to have enough liquid for a refill before each run, thus minimizing the evaporation effect, only three of the six holes - H2 (4x2), CALIB (10x1) and RESIDUAL (6x3.5) - were filled with the radioactive saline solution. The probe was fixed in vertical position over a rotating plastic disk at a heigh of 50 ± 10 µm. At the optimal working point the background rate due to the PMT dark counts was measured to be 0.31 Hz. While a distance plate-probe of 50 µm was used in the first test with liquid Yttrium without any problem, in this test, after the first run, an increase of dark counts was noticed, with values raising from 0.3 to 18 Hz due to a contamination of the probe tip. This effect of tip contamination was noticed also increasing the distance at 100 µm, and was associated with an electrostatic effect of the aluminium (the probe tip in the first liquid Yttrium test was made in PVC). It has to be noticed that in clinical application the probe must be used with a plastic cover to ensure the sterilization, so this effect is expected to be minimized. The probe and the plate were cleaned, even if a residual contamination remained on the disk, especially inside the holes. This contamination, even if uncontrolled, was used to simulate a more realistic situation (in clinical cases a diffuse background due to the uptake of the surrounding healthy tissue is expected). On the contaminated plate, paper spot of 100 µm thickness sopped in Yttrium were placed near the holes (fig. 4.18). BESTA was refilled with the radioactive solution,

72 66 4. Probe design while CALIB was dried. The distance probe-plate was increased to 700 µm to avoid further contamination. A scan over the plate was acquired, and the result is shown in BESTA was easily identified, as most of the spots. The residual contamination of CALIB was enough for its identification. Contamination points out the need of a specific set-up to estimate the probe s answer under tip contamination during surgical procedure (par ) and a new techniques to test the probes properties, that will be discussed in detail in sec Figure View of the plate with the paper spot on it Figure S4 scan over the disk, when a a controlled contamination was inserted. The nominal activity was 11.5 kbq/ml 4.4 ProbeSiPM Another detector, named ProbeSiPM, was developed to test the effect of a different light collection device, replacing the PMT with a SiPM [56]. SiPM is a photon sensitive device built from an avalanche photodiode array (APD) on common Si substrate. Each APD is a small cell, that works Geiger-mode, with dimension ranging from 20 to 100 µm. The cells density can be up to 1000 per mm 2. Each pixel of the array emits a pulse when it detects photons, and the SiPM s output is the sum of the pulse from each pixel. This allows to counts the number of photons detected for each event. SiPM presents several advantages respect to PMT, like a higher quantum efficiency and a higher gain at lower voltage. When SiPM is exposed in an environment with a high flux of particle, the APD recovery time may result limiting. The other disadvantage respect to the PMT is due the optical crosstalk that can occurs in neighbouring pixels. The SiPM was directly coupled in the middle of a disk of polycristalline DpT with a diameter of 10 mm and height of 2.5 mm, removing the optical fibres. The detector head was shielded by the lateral radiation with a torus of black PVC with on top a

73 4.5 Minisipm 67 cover of 100 µm of aluminium. With this direct optical coupling and the use of SiPM, a higher sensitivity to low energy electrons is expected, producing a positive impact on the dose injected to the patient, allowing the possibility to reduce it. The substitution of the PMT with the SiPM obliged to create a new front-end electronics board, that was always based on Arduino Due. In this case the RC circuit was removed and substituted by a pre-amplifier. The process of pulse discrimination and counting remained the same. An internal transformer allowed to drive the SiPM with the correct input voltage when the box was supplied by a portable power pack. The use of a portable power pack reduced considerably the mobility problems of the prototype. In addiction to the tablet, a blinking led and a buzzer allowed to obtain real time and easy to comprehend feedback about the probe counting. With a gain of 27 V and a threshold of 9 mv, dark count rate resulted less than 1 Hz, and this was fixed as probe working point. A scan over the point like source of 90 Sr in air was made. The probe was fixed on a linear actuator at the counts acquired with an acquisition time of 20s. The result of the scan is shown in fig As expected, there was an increment in the rate acquired of a factor 2 respect to Probe4. Figure Resolution of ProbeSiPM over a point like source of Strontium 4.5 Minisipm Exploiting the information acquired with the previous prototypes another probe, called Minisipm, was built, aimed to reach the best possible configuration.

74 68 4. Probe design The core of this probe had the same shielding and active volume dimensions (diameter of 5.1 mm and height of 3 mm) of Probe4, and was directly coupled to a SiPM (like in case of ProbeSiPM). The probe, had an interchangeable head so it was possible to use disks of poly and mono DpT. The differences about the two heads were compared making scans over the point like and diffuse sources of 90 Sr. The shape of the profiles acquired was the same for both the disks, even if in case of mono crystalline DpT an increase in rate of a 15% was observed, as shown in fig.4.21 (point-like source) and in fig (diffuse source). Figure Minisipm s scans over the point like source of 90 Sr with a head of poly/mono DpT (orange/blue dots) Figure Minisipm s scans over the diffuse source of 90 Sr with a head of poly/mono DpT (orange/blue dots). Differences in rate were noticed but the reconstructed profile was the same A direct comparison between Minisipm and Probe4 is reported in fig Being the active material the same (polycristalline DpT) and with the same dimensions, the difference in counts are due only to the optical coupling. The higher light collection efficiency resulted in a lower energy cut, in fact the detection depth (determined as for Probe 4 as explained in sec ) for this probe resulted to be 9.3 mm, as shown in fig The device energy threshold estimated with simulation resulted to be 250 kev. Minisipm looks a good candidate to start with the development of clinical grade β counting probe.

75 4.5 Minisipm 69 Figure Comparison between Misipm and Probe4. In this scan over the point like source is evident the effect of the new light collection Figure Counts acquired with Minisipm in water, statistics of 3ks/point for blue point, acquisition time for the dark count 20k s

76

77 Chapter 5 Evaluation of detector performances In chapter 4 different detectors suitable for β -RGS were presented together with the laboratory tests made to evaluate their performances. In the first part of this chapter further studies made to reproduce realistic environment using wide area solid phantoms are reported. In the second part, using simulation, detector characteristics were combined with the information obtained from PET and SPECT scans (see sec. 2) to estimate probe performances on real clinical cases. At the end, the results of the first preclinical test are presented and discussed. 5.1 Development of the phantoms A critical aspect in the test of the prototypes for the development of the new RGS technique is the difficulty to perform realistic studies before the preclinical tests. To this aim, specific phantoms were designed to reproduce finite size remnants, with activities and topologies chosen to simulate those expected in real clinical cases. Phantoms were also useful to go through different kinds of feedback to evaluate the best assist for the surgeons during the operation Validation of the phantoms Phantoms were developed to simulated a situation closest as possible to the clinical ones (small tumour residual embedded into a larger area of non tumour tissue) in controlled and reproducible experiments. To avoid uncontrolled contamination (see sec ) a material able to encapsulate the liquid radiotracer had to be adopted. The solution selected was a sponge material, the commercially available Wettex Classic by Vileda. This sponge was composed of 65% cellulose and 35% cotton fibres (density 0.14 g/cm 3 ) and was packed as 20x20 cm 2 wide and 2.5 mm thick dry sheets. The choice of this sponge was driven by its high water absorbability of 2.8 l/m 2, (the sponge is able to absorb up to 15 times its own weight), the possibility to easily obtain sharp 71

78 72 5. Evaluation of detector performances and precise cut even on millimetric samples and finally, the capability to regain its original dimensions after a wetting-drying cycle (when wet, the phantoms volume had an increase of 50%). A validation test, to evaluate the fitness of the material for the purpose and establish the procedure of the bathing process, was set. The possibilities to obtain the desired concentrations of activities, cut different shapes and explore different patters were investigated. The radiotracer, extracted with a standard fine insulin syringe from a shielded container, was diluted with distilled water to obtain two different concentrations of activities, 10 kbq/ml (activity Blue) and 5 kbq/ml (activity Yellow), values referred at the moment of the shipping. Cylindrical phantoms with a diameter of 20 mm and a heigh of 2.5 mm were cut, and each was bathed with 0.7 ml of solution, an amount equivalent to the 90% of the phantom volume. This quantity of solution was dropped in a small box, then the phantom was bathed using a pair of tweezers. To obtain an higher uptake uniformity, each phantom was rotated to bath both the sides (the bathing process is shown in fig. 5.1). All these steps were made inside a glove box, a clean and radio-protected environment, as shown in fig The walls of the glove box were made in 10 mm thick metacrilate (PMMA), enough to stop the electrons and at the same time ensuring a perfect visibility of the experimental area. Phantoms were dried for 90 minutes using a home made dryer. The oven consisted of a parallelepiped aluminium body with a grid where phantoms were left to dry. The structure was warmed with four heating resistances, and the uniformity of the heat distribution was assured by a small ventilator at its bottom, as shown in fig After the drying process, the activity of each phantom was measured using a reference detector (called calibrator) to have an independent intercalibration of the samples activity. The calibrator consisted of a plastic scintillator coupled with a PM, encapsulated into a black box with a 5 µm thick mylar window. Before the data taking, calibrator was tuned using a point source of 90 Sr with known activity (370 Bq). Figure 5.1. The bathing process: extraction of the radiotracer from the shielded container with a syringe; dropping of 0.7 ml of saline solution inside a small container; bathing of the phantom using a tweezers

79 5.1 Development of the phantoms 73 Figure 5.2. A view of the glove box, the radio-protected environment in which the tests were made Figure 5.3. View of the inside of the glove box to show the home made dryer and the calibrator The samples activity was monitored with the calibrator for the full period of test (10 days). Rates decreased following the Yttrium decay law confirming the trapping inside the sponge of the isotopes. The achievement of the planned dilutions was verified. In fig. 5.4 are reported the counts for second of the first day of data taking for each of the 10 phantoms prepared for the test. Differences between samples were lower than 5%. The level of homogeneity found had to be compared with the usual uncertainty of uncalibrated radioactive sources of 20% [61]. As shown in the plot the ratio between the obtained activity was different from the expected one (1:2). This was related to some pipetting problems with the syringe, for the presence of occasional air bubbles when the solutions was picked up. A not completely uniform tracer uptake among sample volumes was observed. Despite some variations due to systematic effects of samples preparation the mean difference between the up and down sides was in the order of 5%. The bathing process was accounted for the up-down effect, being the down side, the one that showed an higher uptake, always bathed first. Considering the results obtained and the handiness of the samples during the procedure, the techniques proved to have a lot of potentials, and further studies were made. Phantoms are expected to give the possibility to produce both wet or dry surfaces depending on the scope of the test. Dry phantoms may be used as sealed sources to test the physics detector performances, like probe resolution and profile reconstruction. Wet surfaces may be used to estimate the effect of tip contamination, or to evaluate probe performances in a more realistic environment Realization of real topologies with phantoms A second bunch of phantoms was created to simulate some real patterns and TNR may show up during surgery, aiming to estimate the realistic probe performances. Standing the handiness of the phantoms, in this run different profiles were cut to be

80 74 5. Evaluation of detector performances Figure 5.4. Rate acquired with the calibrator for the ten phantoms. For each phantom are reported the counts per second for the up and down side. Blue activity 10 kbq/ml, Yellow activity 5 kbq/ml combined in complicated geometries. The following shapes were made: - Spot, a small disk with 5 mm diameter - Disk, cylinder with 20 mm diameter - Torus, with inner diameter of 5 mm and outer of 20 mm for all the phantoms the height was 2.5 mm. The typical assembly with this technique was obtained inserting a spot into a torus to simulate a 0.05 ml tumour residual surrounded by healthy tissue. A disk with an appropriate activity was eventually used to reproduce the presence of further healthy tissues above or under the hot spot. A TNR of 10:1 (the lower ratio expected in clinical cases of meningiomas) between the tumour and the surrounding healthy tissue was realized drenching the phantoms with the properly diluted 90 Y solution. Not activated phantoms were used to simulate necrotic areas, dead tissue that due to the lower metabolism do not show significantly uptake of tracer. In this way, different configurations were created, as shown in fig The phantoms were dried, and, before forming any assembly, the individual activity of each phantom was measured with the calibrator. Scans over the described assemblies were performed by fixing Probe4 (see section 4.3) to a XZ motorized system. The X linear actuator run the probe over the phantoms in 1 mm steps, with a measurement time of 10 s per position, while the Z actuator was used to set the tip to surface distance with a precision of 1.5 µm. The starting probe-phantom distance was fixed to 100 µm. Probe motion and data acquisition were automated, synchronised and driven by a LabVIEW program.

81 5.1 Development of the phantoms 75 Figure 5.5. Different topology created with phantoms technique, to test the discovery potential of the probe. These assemblies reproduced schematically expected configurations of tumour locations. Colour code: White non-active phantom; Celeste: low active phantom; Red: high active phantom Problems related to air bubbles during radiotracer pick up were solved replacing the syringe with two adjustable-volume calibrated micropipettes (with capacity of or µl). To reduce inhomogeneity among the phantoms, the saturation level was increased up to 100% of the volume. This did not bring the expected results and up-down inhomogeneity reached the 16% in the larger samples (disks and torus), while the differences between samples were in the order of 10%. In spots, the smallest samples, it was not possible to measure the up down difference (standing the impossibility to differently mark the two sides), but an higher uniformity among them was reached, since half of the sample showed inhomogeneity lower than 5%. Local disuniformity, if any, was not critical being these samples smaller than the probe dimension. In fig. 5.6 the reconstructed profiles on different configurations are shown. The scans were acquired at different times, so, for a direct comparison between them, the counts acquired were corrected for the Yttrium decay time, normalizing all the rates to a reference time t 0. Tip contamination was not spotted, and the dark counts of the device resulted lower than 1 Hz. To characterize probe s performance, as parameter of interest was chosen the discovery potential, defined as the threshold value that the probe s counts had to overcome to identify a region of interest. This values, reported on the plots as a line, was defined (offline) fixing the false positive probability (see sec ) less than 1%, lasting one or ten seconds for position. The rate acquired on an isolated disk with the same uptake of the healthy tissue (21 Hz) was considered as benchmark to compute this false positive probability. On all the topologies, the region of interest selected by the probe was clearly over the phantoms with higher activities. This happened also in the worst case, hidden and in deep tumour. These scans were redone on the same configurations increasing the probe-phantom distance to 2 cm, and also in this situation, all the spots were identified.

82 76 5. Evaluation of detector performances Figure 5.6. The horizontal lines indicate the minimum rate needed to ensure a false positive probability < 1% with a measurement lasting 1 or 10 s for point. Below the figure is reported a scheme of the phantoms topology. Colours are associated with activity: Celeste indicates an area with the activity of background (healthy tissue), while red an area with 10 times more activities (tumour). The unfilled area corresponds to non-active regions

83 5.1 Development of the phantoms Minimum detectable tumour residual These tests were aimed to evaluate probe discovery power in a "dynamic" situation, where the tumour mass is progressively reduced by surgical removal. For this purpose, parallelepiped shaped phantoms were cut to assemble a different phantoms topology (fig. 5.7). The starting configuration consisted in a long strip, 55.5 mm x 10 mm, cut in seven tiles, five of them (the inner ones) charged with high activity, while the other two (on the bound) with low activity. A TNR of 10:1 was used. A scan over the whole strip was repeated reducing the tumoural area by removing a tile at a time starting from the wider one (6 mm). At each iteration, the assembly was packed again to maintain the tumour area enclosed in the healthy tissues. In this way the evolution of Probe4 answer was evaluated, aiming at the identification of the minimum detectable residual. Figure 5.7. Left: Scheme of the phantom prepared to simulate the progress of a surgical removal of a tumour. The red strip of 5 tiles of different sizes and charged at the tumour uptake was surrounded by two blue tiles, charged with ten times less activity. A sequence of scans was performed removing a tumour tiles at each iteration. Right: Photo of the laboratory test. The blue tile, 2.5 mm long, was charged with high activity while the two green tiles that surrounded it, 20 mm and 15 mm long, were charged with lower activity In fig. 5.8 are reported four of the profiles acquired named, a, b, c and d. Plot a was obtained with a scan over starting configuration, composed by a total tumour area of 20.5 x 10 mm 2 surrounded by two tiles with low activity. As shown in the plot, when the probe reached the tumoural area, higher rates values were acquired, allowing its identification. The shape b represents the response of the probe when the tumour thickness was reduced removing three tiles (6mm, 5mm, and 4 mm). The rates over the tumour decreased, since the total active mass was reduced, but still the tumour margins were clearly identified. The detector answer on the smallest residual tested in this measure is reported in plot c. It was obtained reducing by 5 mm the width of the 2.5 mm long phantom, reducing the volume to 0.03 ml, one third than the volume benchmark of 0.1 ml. Finally, the trend d is the profile acquired on the background tiles, when all the high activity tiles are removed. This is the expected configuration after a complete resection, when no more peaks are localized.

84 78 5. Evaluation of detector performances (a) (b) (c) (d) Figure 5.8. Scans made to identify the smallest detectable tumour: a) Starting configuration, with a total tumour area of 20.5 x 10 mm 2 surrounded by two tiles with low activity. The probe movement was from right to left. The rate counts started from zero, since the probe was outside the scanning area, then increased approaching the healthy tissue. Over the 20 mm long tiles, the rates reached a plateau of 50 Hz, that represented the background level of this measure (equivalent to the uptake of the healthy tissue). Then the rates increased up again moving over the tumoural areas, and reached another plateau at about 500 Hz, lasting as much as the probe sensitive area was fully illuminated by the tumour area. As soon as the sensitive detector exited the last tumour tile the rate went back to that of the healthy tissue. b) Situation on a total tumour area of 5.5 x 10 mm 2, surrounded by two tiles with low activity. c) Situation on the smallest sample, obtained cutting in half the 2.5 mm long sample, reaching a tumour area of 2.5 x 5 mm 2, surrounded by two tiles with low activity. d) Final configuration, were only the background tiles remained

85 5.1 Development of the phantoms 79 Tip contamination The previous scans were performed in a well known configuration, whereas in the real application some perturbations could be expected as, for example, the contamination of the probe tip due to the contact with the wet active tissue. To evaluate this effect, the scan c, reported in fig. 5.8 was used. Each point of the initial profile, p x, was replaced by a new random value newp x = P(p x ) + P(p bk ), where P(p x ) was a random value extracted by the Poisson distribution with mean value p x and P(p bk ) was a random value extracted by the Poisson distribution with mean value p bk. P bk, was a random value, generated in the range 0-100, used to simulate the tip contamination. The value of p bk was added also to the uptake of healthy tissue (extracted from the plateau on the right of the initial distribution) to correctly compute the false positive probability. In fig. 5.9 some scans are reported. Even in case of not physical tip contamination (100 Hz), the probe was able to correctly identify the active spot. The same procedure was repeated reducing the injected activity, rescaling the starting activity for Yttrium decay law, up to 1/10 of the initial value. This study led to the conclusion that an eventual contamination would not be a problem if monitored with a frequent background calibration during the operation. Figure 5.9. Left: Same activity of the starting value, background added 100 Hz. Center: 1/3 of the initial activity, background added 87 Hz. Right: 1/10 of the initial activity, background added 96 Hz. In all the plots the green areas include the values below the threshold, considering an acquisition time of 10 s/point, while the red areas indicate the position of interest identified by the probe Spots identification All the previous tests were made with an automated movement and acquisition system on a well known and flat geometry. A further test to evaluate the handiness of the tool was planned. In this test, an operator had to discover the hot spots, using Probe4 and without time limitations. Thirty-six wet spots were inserted into a plasticine matrix as shown in fig Six of them were activated with high activity, while the others were bathed in pure distilled water. To test at the same time the handiness of the tool, the matrix geometry did not allow an easy approach with the probe to the phantoms. Two different probe configurations were set. In the first the electronics threshold of

86 80 5. Evaluation of detector performances the probe was lowered, increasing both efficiency and dark count. In the second one the optimal working point, found in sec was used. This measure was expected to give indication on which parameter is more relevant for the operator between the S/N and the efficiency. Two different operators, operator A and operator B, used the probe to identify the hot spots. They did not know the number and position of the activate phantoms. The operator A, equipped with the probe in the first configuration, identified 6 spots as tumoural (the red points), but was not sure about 3 samples (the yellow points). The operator B, equipped with the probe in the second configuration, succeeded to identify all and only the hot spots (located in the green squares). In fig the results obtained by two different operators are reported. As shown in the same picture, both of them used an external table to read the rate. The output of this test enforced the importance of the signal to noise ratio as parameter to estimate probe performance. The downside revealed by the experiment was related to the tablet s acquisition time, since rate resulted unsuitable for a real time answer. To compute the rate, the system opens a window and counts the number of events into it. Then this number is sent to the tablet. This means that the number shown on the display is referred to the events happened in the previous time window. To evaluate how to reduce this latency time, an evolution of the test was made, to investigate the effect of different feedback. Figure Left: The plasticine matrix filled with phantoms and the set-up used for the tests. Right: scheme of the matrix and answers of operators A and B (green: positions of the hot spots, yellow: spots identified as possible tumour, red: spots clearly identified as tumoural) Human feedback More important than the pure performance of this kind of device is the way the information it collects are transmitted in real time to the surgeon in a clear and non ambiguous way. The probe is equipped with different way to express the counts acquired. The effectiveness of these different feedbacks was verified with a test.

87 5.1 Development of the phantoms 81 Twenty-five dried phantoms were randomly inserted into a plastic matrix (5x5) (figs and 5.12). The phantoms consisted of a spot inserted into a torus. The number of phantoms for each different combination of activity considered are reported in the following list: - 5 with spot uptake 0 and torus uptake 0-5 with spot uptake 1 and torus uptake 1-5 with spot uptake 10 and torus uptake 0-10 with spot uptake 10 and torus uptake 1 A yellow led driven by a microprocessor (Arduino mega board [62]) was fixed over each position. The software chose randomly one led turning it up for a fixed time and then turning it off. Different time windows were tested, in the range 1-5 s. The led allowed to investigate the mean time that effectively an operator needs for the interpretation of the device s answer and enable to avoid operator s memory effect. The testers were equipped with different feedbacks, numeric (the tablet), visual (a blinking led) and acoustic (a buzzer). As reference, before the test, the rate over a phantom with the same uptake of the healthy tissue was acquired. After the acquisition of this information, the test started. While the led was on, the human testers had to decide if the spot represented a hot spot or not. The process went on until all positions, avoiding repetitions, were checked. The led and the buzzer proved useful for a faster answer, even if no operator was able to take a decision in less than 2-3 s. This was evaluated as the minimum time that an operator needed in the test conditions to identify the selected spot and evaluate the feedback. Figure The board with the phantoms on it. The colour of the different sponges are meaningless Figure Position of each phantom. The colors are related to the activity in this way, red activity ten, blue activity one and white no activity

88 82 5. Evaluation of detector performances 5.2 Simulation Before using this detector on the patients, a further engineering of the prototype is mandatory. In fact, even if during the developing process aspects related to the protection of the patients were considered (e.g. use of sterilised materials, isolation to prevent current leakage) components failure is unpredictable and a certain degree of risk is unavoidable. For this reason, before an effective use of any new medical device, there is the necessity of a certification of the risk assessment that can be done only by dedicated companies. In this "limbo", without certification, to estimate the detector performances without endangering the safety of the patients, simulation plays a key role. In the absence of human data, simulation tests are the most reliable means of accurately evaluating the techniques potentials to submit a certification process Monte Carlo Monte Carlo methods (MC) refers to any method which solves a problem using random generators. MC is particularly useful to obtain numerical results when the problems are too complicated to solve analytically. Today is a standard tool in several areas of research, particularly in physics. Starting from the basic laws of the process, the results of different situations can be obtained even without doing specific experiments. FLUKA (FLUktuirende KAskade) was used among the different MC packages, since it was specifically designed for the simulation of interaction and transport of particles and nuclei in the matter. The software is sponsored and copyrighted by INFN and CERN [63] [64]. A tuning of MC on well known situations was necessary, so it was used to reproduce the laboratory tests results. In this way, free parameters of the simulations (e.g. DpT characteristics, reflectivity of the materials, efficiency of the device) were fixed. The conversion factor between the simulated deposited energy and the electronic signal was also estimated in this way. In reality, the probe had a count when the electronic signal overcame the threshold. This was reproduced in the simulation, where, when the deposited energy was above the threshold, the probe was considered to had issued a count. After this tuning, confident of the output of the simulations, MC was used to estimate probe expected performance in clinical cases False positive and false negative probability MC estimated the expected rates on both tumours and healthy tissue (respectively ν T and ν NT ) but these values refers to decay processes. An higher value of ν T respect to ν NT does not automatically mean that the probe is able to clearly identify the tumour with respect to the surrounding tissue, since this values represent the mean

89 5.2 Simulation 83 value of a Poisson distribution. To correctly take into account statistical fluctuations, the probe performances were evaluated considering the probability of False Positives (FP) and False Negatives (FN). An event is referred as FP when the result of the test indicates the presence of the disease, even if the sample is healthy. On the contrary, FN is defined as an event in which the result of the test does not indicate the presence of the disease, even if the sample is ill. Referring to RGS, FP indicate the number of times in which healthy tissue is removed, increasing the overall impact of the surgery, whereas FN indicates the number of time in which tumour s remnants are missed, thus increasing the possibility of recurrency. Being statistical fluctuations, set ν T and ν NT, FN e FP depend only the time of integration for the sample. The minimum time (t min ) that a surgeon needs to spend on a sample to evaluate whether it is healthy or not was chosen as the parameter to evaluate probe performances. Given ν T and ν NT, a specific macro was written to determine t min, defined as the minimum time for which exists a value of N thr such that FN< 5% and FP< 1%. N thr indicates the threshold value over which the probe identifies an area of interest. An example is reported in fig to clarify this definition. In case of ν T =10 Hz and ν NT =3 Hz with an acquisition time of 1s, there is a wide area of overlap between the two distributions. This affects the value of N thr. In fact to obtain a FP probability lower than 1% a N thr higher than 8 Hz is requested, and respectively to obtain a FN probability lower than 5% a N thr lower than 5 Hz is requested. Since a common value of N thr can not be find in this hypothetical case, the probe can not be used lasting only one second for position. As the plot on the right shows, waiting 5 s the distribution fluctuation are reduced, and there is no overlap over the two distribution. This means that a probe answer higher than 6 Hz identifies, with the correct probability, a tumour. Figure Probability distributions in case of two signals of 3 and 10 Hz, lasting 1/5 s (left/right) Mathematically, FP e FN for an acquisition time of the probe (t probe ) could be

90 84 5. Evaluation of detector performances computed with the following equations: N thr 1 F P = 1 P νnt t probe (N) (5.1) F N = N=0 N thr 1 N=0 P νt t probe (N) (5.2) where P ν (N) indicates the Poisson probability of having N if the mean is µ. RGS can be practical only if, when administering the reference activity (3 MBq/kg), the time t min is not significantly longer than 1 s, a reasonable time elapse in the surgical environment. Otherwise, an increase of activity would be needed. On the contrary, if t min is shorter than 1 s there would be margins to reduce the administered activity Performance on clinical cases As aforementioned in sec 2.3, statistic studies were presented to evaluate the DOTA- TOC uptake in brain and abdomen tumours. From the SUVs estimated, the specific activity in tumour and non-tumour at the time of the surgery for each patient were computed assuming a radiotracer injection 24 hours before the intervention. The activities were corrected only for the decay time and not for the biological wash-out of the organs. Indeed, as shown, washout is faster in the healthy tissues than in the tumour, leading to a underestimation of the TNR, so the conclusions of this discussion are conservative (especially the one relatives to brain tumours). These activities were then converted in probe signal rates using FLUKA. The smallest volume detectable by a PET scan corresponds to 0.1 ml. These are indeed the dimension of a typical residual that has to be identified by the probe. In the simulation, the equivalent tumour mass was represented by a cylinder with a 3 mm radius and a 3.5 mm height. The tumour region is assumed to have a specific activity µ T of 90 Y. The residual was surrounded by an extended region with a lower uptake µ NT (fig. 5.14). To estimate the rates in case of healthy tissue, the activity of the inner cylinder was set equal to µ NT. In the simulation, the probe was in direct contact with the tissue. In particular in this simulation, the probe used to estimate the performance of β -RGS was Probe4 (see section 4.3). Even if other prototypes showed an higher efficiency, this model was chosen due the presence of optical fibres. This is an important factor because allows to locate the HV far from the patients, decreasing the potential risk for patient s safety. The results are shown separately for meningiomas and gliomas and NETs. For each patient are reported the expected rates on lesion ν T, healthy tissue ν NT and the t min needed to identify a 0.1 ml residual administering an activity of A ref =3 MBq/kg (the dose used for a PET scan). In case of brain tumours, the minimum activity that needs to be administered to have t min =1 s is also reported.

91 5.2 Simulation 85 Figure Simulation set-up. A tumour residual (red) surrounded by a healthy tissue (pink) with a lower uptake. The probe consists of a cylinder of DpT (white) surrounded by a PVC shield (black). In case of meningiomas, the results proved that the technique would be effective also administering less than 3 MBq/kg, as shown in tab In case of gliomas, an activity of 3 MBq/kg was not enough to identify the lesions in 1s, since the probe requires up to 5/6 s as shown in tab It has to be reminded that SUV in case of glioma were estimated with a conservative approach, moreover glioma patients considered in this study had undergone previous treatments that can have decrease the expression of receptors. In case of NETs, the SUVs in the lesions were particularly high. Therefore the numbers reported in tab. 5.3 were obtained considering an administration of only 1 MBq/kg (one third respect to the other cases). It has to be noticed that A MAX used to estimated the SUV values was computed considering the activity of the bulk of the tumour whereas RGS is expected to be used on the margin that typically showed a lower uptake. The results obtained were very promising, suggesting a good chance of application of this technique in the class of the tumours studied.

92 86 5. Evaluation of detector performances Patient ID N les ν T ν NT t min A min Hz Hz s MBq/kg M M M M M M M M M M M Table 5.1. Number of lesions (N les ), signals rate ν T and non-tumour rate ν NT expected on probe and minimum time t min needed to identify a 0.1 ml residual. These numbers were evaluated considering the administration of the reference activity of A ref = 3 MBq/kg. A min refers to the minimum activity that needs to be administered to have t min = 1 s

93 5.2 Simulation 87 Patient ID ν T ν NT t min A min Hz Hz s MBq/kg GB GB GB GB GB GB GB GB >10 - GB GB >10 - GB GB Table 5.2. Signals rate ν T and non-tumour rate ν NT expected on probe and minimum time t min needed to identify a 0.1 ml residual. These numbers were evaluated considering the administration of the reference activity of A ref = 3 MBq/kg. A min refers to the minimum activity that needs to be administered to have t min = 1 s

94 88 5. Evaluation of detector performances Patient ID N les ν T ν NT t min Hz Hz s N N N N N N N N N N N N N N N Table 5.3. Number of lesions (N les ), signals rate ν T and non-tumour rate ν NT expected on probe and minimum time t min needed to identify a 0.1 ml residual. These numbers were evaluated considering the administration of the reference activity of A ref = 1 MBq/kg

95 5.3 Ex-vivo specimens Ex-vivo specimens The current probe prototype is not certified according to EC regulations, so its direct use in the surgical field is not possible to achieve. Ex-vivo experiments allow to test the probe on real tissues bypassing this limitation. Ex-vivo tests refer to measurements done on tissue extracted from an organism with minimum alterations of natural conditions. This type of measures enables experimentation in controlled situations avoiding, at the same time, any risk to the patient. The first test on ex vivo specimens was carried out in collaboration with the Istituto Europeo di Oncologia at the Istituto Neurologico Carlo Besta, in Milan, on October 8 th 2015, on a patient affected by meningioma. This study was approved by the Ethics Committee (that considering the scientific value and the ethic of the experiment justified the radiopharmaceutical administration, not already foreseen), and the patient (first of the five approved for this experimentation) gave written informed consent to participate in the clinical research. The procedure, assessed in collaboration with the medical team, was organized considering the safety of the patient as the primary objective. A PET imaging exam was performed on the patient 3 weeks before the operation. The information of this scan was used to quantify the receptivity to the tracer of the patient s tumour with respect to the healthy tissue. The patient showed a non particularly high SUV value (the SUV values were: max=4.11; mean=3.1; min=1.9). Considering the administrable activity on the basis of the approved protocol, the patient was injected 24h before the operation with 255 MBq of Y90-DOTATOC (4 MBq/Kg, considering the weight of the patient of 64 Kg), corresponding to a mean tissue radioactivity concentration of 12.4 kbq/ml. Using this value in the simulation, the expected rate on the bulk (reproduced as a sphere of 5.5 cm 3 ) and on one residual (sphere of 0.3 cm 3 ) resulted respectively 128 and 83 Hz. One hour before the surgery, the probe counts on the skin of the patient in different positions were acquired, to confirm the insensitivity of the device to long range radiation and to estimate the rates on healthy tissues with low uptake of tracer. Then the patient underwent the surgery. During the surgery, as stated by the protocol, tumour samples and a small portion of healthy tissue (from the dura) were extracted and each sample was located in a specific marked box. The practice to remove some healthy tissue falls within the cost/benefit considerations. To reduce the possibility of recurrence it is preferable to remove a surrounding healthy region near the lesion. It has to be noticed that the test did not interfere with the routine surgery and no decision was taken by the surgeons on the basis of the responses of the probe in this testing phase. The extracted samples where sent out from the operating room, in a dedicated room were the counting rate of the samples was measured with the probe prototypes (Minisipm described in sec. 4.5). After these measures, the samples were submitted to the anatomo-pathologist for the morphological characterization of the tissues.

96 90 5. Evaluation of detector performances The counting rate acquired by the probe on the skin of the patient are reported in tab The counting rates were estimated placing the probe in contact with the skin in 4 different positions with an acquisition time of 10s. Two positions at the maximum distance from the lesion (the right arm and the right foot) and two almost close to the lesion were investigated (Head 1 was chosen on the head but far from the lesion, while Head 2 was chosen in the nearest point to the meningioma). The dark counts of the device were acquired before and during the run, and resulted always lower than 1 Hz. These measures confirmed the insensitivity of the probe to Bremsstrahlung induced by electrons in the body and gave an idea of the uptake of the tracer far from the lesion (the expected background for the measure). Position Rate [Hz] Right arm 6 Right foot 11 Head 1 15 Head 2 17 Table 5.4. Rates acquired on the skin of the patient before the surgery, waiting 10s at positions. Head 1 refer to a position on the head far from the expected position of meningioma, while Head 2 was the closest position to the meningioma on the head The set-up and the procedure organized to measure the samples of tissue are shown in fig The set-up consisted of a plane covered with graph paper under the field of view of a camera to record all the measure steps. The graph paper allowed an offline estimate of the dimension of the surface of the samples. Two additional led lights set the proper illumination. Each sample was extracted from its box, weighted with a digital scale, and inserted into a glass container (to avoid liquid drop) located under the camera. A thin polyethylene film was lied on the sample to avoid the direct contact with the probe head (to prevent tip contamination as explained in sec ). Two images recorded with the camera are reported in fig. 5.16, to give an idea of the shape and dimension of the bulk of the tumour and of a possible residual. After the procedure each sample was bathed in saline solution and placed in its box. The preliminary results of these measures are reported in tab For each sample the weight, the site of resection, the surgeon assessment and the counts acquired with the probe (10s for each measure) are reported. Due to the complex shape of the samples the activity was measured touching the samples in different places. The highest counts were acquired over the tumour mass, while on the dura (probably healthy) the rate was significantly lower. These preliminary results were consistent with the expected behaviour.

97 5.3 Ex-vivo specimens 91 Figure The set up used to analyse the ex-vivo samples. The probe electronics was located inside the white box in the first picture, and the rates read by a display located on the same box. The second picture shows a moment of the data taking, when due the slipperiness of the samples, a plastic stick was used to handle them Figure Two of the samples analysed. On the left the meningioma, on the right a different sample (border of the lesion)

98 92 5. Evaluation of detector performances The samples F and G were cut from the bulk of the meningioma (sample D). They had the same uptake of the bulk, but lower rates were acquired. These were related to the reduced dimension of the samples, smaller than the field of view of the probe. A correct comprehension of these data, requires to considered the rates as a function of the volumes of the sample. In any case, the results of the anatomo-pathological measures (not yet available) will define the real nature of the samples examined. The tests was a success, even if a detailed analysis is not yet complete, the preliminary results obtained confirm the expected ones, and it all seems to bring to a direct use in the surgical field. Sample Weight [g] Surgeon rating Probe [Hz] A 0.39 Dura (perhaps not infiltrated) 2,3,7,8 B 0.23 Lesion (upper border) 50, 53 C 0.73 Lesion (lower border) 53,37 D 4.84 Lesion (core) 89,91,106,110,115 E 0.88 Dura medial margin (perhaps infiltrated) 4,3 F 0.21 Meningioma (sample) 21,29,33 G 0.39 Meningioma (sample) 39,42,37 Table 5.5. For each sample is reported its weight, where it was taken and the surgeon evaluation, and the counts acquired with the probe (10s for each measure)

99 Chapter 6 Development of a multichannel probe The development of β probes has been reported in the previous chapters. These radiation detectors were designed to provide a single channel signal, allowing the surgeons to get information about the distribution of a radioactive labelled structure. The acquisition of functional images has been investigated with the aim of a further improvement. In this case the detector must be able to reconstruct 2D images, increasing the number of information provided to the operators. A portable imaging detector will ensure all the advantages of medical imaging being at the same time ease to use. In this chapter the first steps needed to create an imaging probe are reported. 6.1 Design of the imaging probe To reconstruct a medical image, the counts acquired by the detector have to be related with the activity of the underlying tissue. Probing the surface in different points at the same time allows to reconstruct the activity map if the interaction point of the electron on the active material is correctly identified. Considering the constraints in case of a medical portable detector, this can be obtained in two ways: - dividing the active material in a matrix of smaller active volumes (little crystals) each with its read out - using only one crystal, increasing the number of read out at the end of the volume each proposal has its advantages, so both were investigated. In the first design, the spatial resolution is expected to be dominated by the crystals dimension. Being the response the same for all the crystals independently from their position, a spatial linearity is expected in the image reconstruction. 93

100 94 6. Development of a multichannel probe The second model allows a better light collection, avoiding the dead area in the intersection between the crystals, increasing the "filling factor" Pixelated crystal With the aim to test the first solution, the creation of a detector made of a matrix of cubic crystals was hypothesized. The idea consisted of 16 crystals optically isolated on a support structure each coupled with its own readout. In this configuration the image is expected to be reconstructed considering the difference in counts between the crystals. The properties of a single crystal were studied in view of the matrix solution, investigating particularly the most effective way to cover the crystal to prevent optical crosstalk. The use of DpT was avoided, being impossible to machine it in so small cubic pieces with the laboratory tools. A different active material, CsI(Ti) was selected. CsI shows a high light yields ( 54k γ/mev [65]) even if this material has a higher gamma sensitivity. As aforementioned, in case of a use of 90 Y, Bremsstrahlung radiation is expected to be very low, anyway in view of a clinical application, this can impact on the possibility to use other no pure beta minus emitter radio tracers. The crystal used had a cubic shape, with a volume of 2x2x2 mm 3 (the crystal is shown in fig. 6.1). Optical proprieties were not the same on all the crystal surface. Two faces were polish, while the other four were rough. The first aspect investigated was related to the cover of the rough sides from ambient light in view to prevent crosstalk. In a matrix configuration the possibility to realize this thin cover will be a key point. A paint solution was tested, but standing the small dimension and the reduced handiness of the crystal, an uniform cover was impossible to obtain. The following attempt was a casting of black silicon into a Teflon form. A complete adherence between CsI face and Teflon was impossible to obtain, and a significant fraction of cover reached the crystal polish face. A different solution was tested using a 3D printer. A white cylindrical plastic armour was printed to cover the side of the crystal, as shown in fig The diameter of the final compound was 10 mm. Although this shielding was too thick, a printed cover showed the necessary potential required by the final design. Light collection played another important role, that had to be investigated. An optical fibre was glued in the middle of one of the polished faces. Different glues were tested, but none allowed to reach an appropriate mechanical stability, standing the reduced dimension of the contact surface (fibre diameter 1 mm, crystal face 2x2 mm 2 ). To reach a stable configuration the use of fibre was avoided and the crystal was directly coupled with a SiPM using optical grease. In this configuration

101 6.1 Design of the imaging probe 95 alignment played a key role, being the SiPM active area 1x1 mm 2. The signal collected by the SiPM was shaped and amplified with a custom electronics chain of CAEN modules. Using a linear actuator, the crystal was centred over a point source of 90 Sr. The maximum rate acquired was 39.2 ± 0.6 Hz, with a dark counts rate of 1.3 Hz. This result can be compared with the one obtained using Probe1 (see sec. 4.2). Probe1, a prototype with a head on DpT, had a different light collection (optical fibre coupled with a PMT) but a comparable active volume (6 against 8 mm 3 ). On the same source the counts reached 59.0 ± 0.8 Hz. Even if there would be a margin of improvement in case of CsI, further development of this option were abandoned. The handily complication, the impossibility to use optical fibre, the counts obtained were considered a sufficient drawback to move on the single crystal solution. Figure 6.1. Size comparison of the crystal respect to a 1 cent euro coin Figure 6.2. View of the crystal wrapped into its white plastic shield Single crystal Scope of this work was to investigate the potential of the second solution, a detector constituted of a single crystal coupled with many photodetectors. Monocrystalline DpT was selected as active material. The short lambda of DpT was estimated to be enough to contain the transverse dimension of the energy released, allowing the identification of the interaction point. In addiction, as shown in sec. 3.3, the diffusion of the material showed promising results in view of an image reconstruction. To test the hypothesis a DpT disk with a diameter 25 mm and height 2.5 mm was coupled to multianode PMT (HAMAMATSU H7546A [66]). The PMT allowed to read 64 channels at the same time, even if for these measures only 16 of them were activated to reduce the technical difficulty. The optical coupling between the crystal and the PMT was guaranteed by small optical fibres (length 3 mm, diameter 1 mm) inserted into a holed plastic matrix. The holes were drilled in correspondence

102 96 6. Development of a multichannel probe of chosen channels (anode size 2x2 mm 2 ). The reconstruction of an image requires that the same picture is acquired from different positions. Being the electronics to process 16 channels in parallels not available at the moment of the data taking, only one channel could be used. To overcame this limitation, the system was motorized. In this way the selected channel was able to explore the surface. Multianode was fixed inside a black box, while the DpT was inserted in the hollow of a specific designed and printed actuator (fig. 6.3). Over the active volume was inserted a 600 µm copper "butterfly" mask and on top of this compound the diffuse source of 90 Sr (the butterfly mask is shown in fig. 6.4). This copper thickness was enough to stop the electrons with an energy lower than 1.1 MeV. The movement was assured by two linear actuators, that allowed to explore an area of 10 x 10 mm 2. Scope of this measure was a qualitative estimation of the single crystal approach. The butterfly mask well suits this since two hot spots easy to identify are expected, being the contribution under the wings suppressed. Figure D design of the plastic actuator Figure 6.4. The butterfly copper mask used to project an image The first image reconstruct with this approach is shown in fig The scan was made waiting 100s/position, with a step of 0.5 mm in each direction. The two black lines represent the border of the two butterfly wings, while the semicircle indicates the border of the radioactive source active area. The profile of the copper shield is clearly identifiable, even if the picture is not completely symmetrical. During the test, the disk had a crack. It was used the same, being the only one available, although a negative consequence was expected (fig. 6.6 clearly shows the crack of the disk). To evaluate if the shift can be related to the crack, a scan of the surface, removing the copper, was acquired. The result is shown in fig The effect of the fractures are clearly visible, and cause a non uniformity of the crystal answer among the surface.

103 6.2 Isolated spot identification 97 Figure 6.5. First image reconstructed of the copper butterfly shield To compensate this effect, the original map (6.5) was reweighted with the following equation: newz xy = (z xy /w xy ) maxw xy (6.1) where z xy are the counts in the point x,y of the original map, w xy are the counts in the same point x,y of the naked disk map, and maxw xy the maximum value of counts acquired in fig 6.7. As the fig. 6.8 shows, with this procedure, the shape of the copper is easier to identify. The test performed with the cracked disk pointed out the necessity of an image post processing. The reweight was useful but showed some limits. An overestimation of the activity in the border region (X=0, Y=10 in fig. 6.8) is clearly visible. Being this region out of the surface of the active source, lower counts respect to the other points of the maps are expected. The results of this quantitative tests was sufficient to proceed with the development of the single crystal hypothesis. 6.2 Isolated spot identification Qualitative tests to evaluate the possibility of a single spot identification were made. The set-up used was the same of the aforementioned test, but a different mask was used. The copper butterfly was replaced by a 1.2 mm lead thick disk. A hole of 0.7 mm was drilled in it, then the same hole was progressively widened to 1.2 mm, 1.4 mm, 1.6 mm, reaching the final dimension of 2 mm.

104 98 6. Development of a multichannel probe Figure mm monocrystalline DpT cracked used as active volume Figure 6.7. Effect of the crack of disk on the light output Figure 6.8. The image reconstructed of the copper butterfly shield after the reweight

105 6.3 Spots pair identification 99 To correctly take in account the effect of the disk crack, a different acquisition channel was selected to investigate a wider area out of the active surface of the source. This sector was selected as control region, an area were the counts are expect to be very low. The steps of the reconstruction algorithm used to process the image are reported in fig Four plots are shown: a) Top Left: raw image acquired waiting 100 s /position and with a 0.5 mm/step b) Top Right: image of the cracked naked DpT disk c) Bottom Left: Process image of the hole (in this case the one of 1.4 mm) d ) Bottom Right: Gaussian fit of the Y-profile (fixing the x of the hole) of the processed image. To process the raw image, the mean value of the counts for x<1.5 were computed in the plots a and b (respectively mean a and mean b ). The value of each point in plot c was computed as z(c) xy = z(a) xy z(b) xymean a mean b (6.2) The result of this procedure is shown in fig. 6.10, where for each hole is reported the sigma and the max value of the Gaussian profile. All the holes were correctly detected, with a comparable profile. 6.3 Spots pair identification These group of measures was acquired to analyse the ability of the device in the identification of two near spots with different dimensions. This is expect to be a problematic situation in clinical cases when the dimension difference between the two spots are marked, because in this case radiation coming from the greater one can hide the other. The mask used for the previous measures was modified by the addition of another hole drilled at a distance of 3 mm (between the center of the holes). The starting hole had a diameter of 0.7 mm, and also in this case was progressively widened to 1 mm, 1.5 mm and 1.8 mm (the final configuration is shown in fig. 6.11). The same set-up and the same reconstruction image algorithm were used in these measures. The effect of the reweight is shown in fig This procedure failed to identify two near spots when one of them had a diameter lower than 1.8 mm. The maps acquired for the hole of 1 mm and the hole of 1.5 mm are shown in fig In this maps was possible to identify only one spot, associated with the 2 mm hole. When the diameter of second hole reached 1.8 mm two spots were identified (fig. 6.12). These tests proved the potentiality of an image detector made with a single crystal of DpT.

106 Development of a multichannel probe Figure 6.9. Steps for the reconstruction of the image. From the top: raw counts acquired waiting 100 s /position and with a 0.5 mm/step on a holed lead mask (1.4 mm hole diameter); counts on the cracked naked monocrystalline DpT disk; estimated counts after the reweight process; y-profile for x=7.5 Figure Sigma (red squares) and max value (blue dots) of the Gaussian profiles as a function of the hole diameter in a lead mask

107 6.3 Spots pair identification 101 Figure The 1.2 mm thick lead mask used for the test. Two holes were drilled (2 mm diameter and 1.8 mm diameter). The distance between the center of the holes was 3 mm Figure Steps for the reconstruction of the image. From the top: raw counts acquired waiting 100 s /position and with a 0.5 mm/step on a holed lead mask (two holes, one with 1.8 mm diameter and the other with 2 mm diameter); counts on the cracked naked monocrystalline DpT disk; estimated counts after the reweight process; estimated counts after the reweight process with a different graphic option to underline the level surfaces

108 Development of a multichannel probe Figure Reconstructed images in case of two near spot: left 2 mm diameter and 1 mm diameter; right 2 mm diameter and 1.5 mm diameter Even with a not optimized probe, single spots with a diameter up to 0.7 mm were identified. Results are expected to improve replacing the cracked disk with a new one. In addition the availability of the designed electronics could produce a further improvement.