International Atomic Energy Agency
THEMATIC PLAN ON DIAGNOSTIC RADIOLOGY

Vienna, Austria May 2003

TABLE OF CONTENTS
Introduction.2 Problem context2 Disease Context2 The technology Context4 Proposed Programme Strategy.5 Conclusions.11 Action Plan11 Appendix 1: The Agencys TC Programme.13 Appendix 2: Acronyms.14 Appendix 3: Meeting Agenda15 List of Participants.21
INTRODUCTION Problem context The developing and less developed nations of the world are still burdened by the problems of malnutrition and the scourges of infectious diseases, which to a large extent have already been controlled in industrialised countries. AIDS and tuberculosis exact a death toll of 13 million per year in the developing world. It must be stated that in many of these countries life expectancy is not high enough for the diseases, which are common in the industrialised countries to be viewed as important problems. However, as development proceeds in the developing countries, with associated improvement of social conditions, provision of safe water supplies and basic medical treatments, this situation will change. It is well documented that average life expectancy is increasing in most countries. However, within developing countries, longevity is increasing linearly with time with the rate of increase being more dramatic than in industrialised countries. It is estimated that over the next ten years the incidence of the diseases associated with a Western Lifestyle, characterized by a diet, rich in fat, refined carbohydrates and animal protein, combined with low physical activity, will increase by some 50% in the developing world. This represents a looming crisis for healthcare provision, but many developing countries are not cognisant of the scope of the potential health care problems and therefore, for instance, have no comprehensive national cancer control programme in place with the requisite human resources and appropriate financial support. It is this lack of understanding and preparedness that presents an imminent threat to citizens in the developing countries and a major challenge to healthcare providers.
Disease Context Some of the diseases associated with the Western Lifestyle that are expected to increase significantly in incidence in the coming years in developing countries are the following. Cancer Cancer is the second leading cause of death in industrialised countries and is among the three leading causes of death for adults in developing countries. In the year 2000, malignant tumours were responsible for 12% of the nearly 56 million deaths worldwide from all causes. That is more than the percentage of deaths caused by HIV/AIDS, tuberculosis and malaria put together. (UICC, WHO 2003) Like the major infectious diseases, cancer is a public health problem for men and women, for young and old, and for rich and poor alike. Once considered a Western disease, more than 50 percent of the worlds cancer burden in terms of both numbers of cases and deaths already occurs in developing countries. Certainly, the causes and types of cancer will vary in different geographical regions but in most countries, there is hardly a family without a cancer victim. Data from the International Agency for Research in Cancer (IARC) predict that by 2020, regions with traditionally low numbers of cancer deaths could see alarming increases in mortality rates. Regions, including Northern Africa and Western Asia, South America, the Caribbean, and South East Asia, could face increases of over 75% in the number of cancer deaths by 2020 as compared to 2000 (WHO, 2003).
For no other illness is the early and accurate diagnosis and proper localisation of disease extent more important than in the case of cancer, and yet, in developing countries, 80 percent of cancer victims already have late stage incurable tumours when they are diagnosed. Diagnostic imaging of all types plays a crucial role in the diagnosis and localization of almost all types of cancer. It is also integral to the management of these patients as a means of monitoring the evolution of the tumour and the effect of treatment. Cardiovascular diseases Every year 12 million people worldwide die from cardiovascular diseases according to WHO, with most of them in developing countries. The main risk factors are high blood pressure, high cholesterol, and smoking, which are traditionally linked with affluent lifestyles but are now being seen in middle-income groups in poorer countries. Not only will the incidence of myocardial infarctions rise and be one of the leading causes of mortality, but also strokes (brain attacks) cause severe morbidity in the populations of developing countries, not to mention the loss of lower limbs from peripheral arterial disease. About half of all the strokes in the world occur in Asia. (Murray CJL, Lopez D. Global comparative assessment in the health sector, WHO 1994). In Sub-Saharan Africa and the Caribbean, the case fatality rates for strokes are 1 to 3 fold higher than in industrialised countries. In the past catheter angiography has been one of the primary diagnostic tools in the investigation of these diseases. However, it is increasingly being replaced by other imaging modalities such as CT or MR angiography, which are not only less invasive and less expensive but require less operator skill. Contrast enhanced MR and the latest generation of multidetector CT are now getting to the point of being able to image a large proportion of the coronary arteries and are already replacing catheter angiography for assessment of renal, aortic (thoracic and abdominal) and the lower limbs vessels, as well as the carotids and the intracranial vessels. In addition, the ability of MRI to quantify ventricular ejection fractions, shunt sizes and the perfusion and viability of myocardium is acceptable in daily practice. Imaging is also being used to identify those patients who have early signs of atherosclerotic disease by either measuring coronary artery calcium, intimal medial wall thickness of the carotids or by directly visualising the atherosclerotic plaque in the carotids or coronary arteries and then following them to determine if there has been response to therapy being instituted. Although catheter angiography is being replaced in the diagnosis of cardiovascular diseases, interventional angiographic procedures still play a major role in the treatment of these diseases. Osteoporosis The WHO has listed osteoporosis as one of the most important chronic diseases today, with a high socio-economic impact. Worldwide, 200 million women may be affected, with at least one in every three women over the age of 50 suffering from an osteoporosis fracture in their lifetime. The most common fractures associated with osteoporosis are fractures of the hip and the vertebrae. It is estimated that hip fractures alone cost about $10 billion per year in the United States. With the predicted increase in the number of older people worldwide, the number of hip fractures could rise from 1.7 million in 1990 to 6.3 million by 2050. It is estimated that 71% of these fractures will occur in developing countries. The development of newer drugs to treat patients with osteoporosis necessitates that those at risk of disease are diagnosed early before they suffer fractures, which in the elderly often result in early death

from complications such as pneumonia. There are numerous radiological methods available for detecting osteoporosis from dual energy absorptiometry (DEXA), quantitative ultrasound (QUS) to quantitative CT (QCT), and these are also used to determine response to treatment. Multi-organ Trauma Multi-organ trauma, which is one of the commonest causes of death in young adults in industrialised countries, is becoming an increasing problem in developing countries. Armed conflicts result in multi-organ trauma to patients of all ages. In South East Asia, motor vehicle accidents are the commonest cause of mortality of young adults (reference) and this has been showing a steady increase. Although these diseases characteristic of the industrialised lifestyle, are dramatically increasing in importance in developing countries, at present, the most common illnesses requiring diagnostic radiology in developing countries are simple limb fractures and diseases of the lungs caused by infections such as tuberculosis. The technology context Over the last several decades dramatic technological advances in medical imaging have placed imaging at the centre of patient management. In the absence of proper diagnosis no rational treatment can be provided and therefore the outcomes expected from any form of treatment would be far from satisfactory. Medical imaging (which is the creation of images of internal structures of the body using a variety of technologies such as x-rays, ultrasound, CT, MRI, nuclear medicine) plays a key role in the diagnosis of countless diseases from infection to infarction, from testicular torsion to tumours and from osteoporosis to osteogenesis imperfecta. There is scarcely a disease process in which diagnostic imaging techniques have no place. Medical imaging uses a variety of technologies such as x-rays, ultrasound, CT, MRI and nuclear medicine. X-rays, CT and nuclear medicine all involve ionising radiation. Although ionising radiation is not used in MRI, it is a technique using the nuclear spin of protons to probe and visualize the chemical structure of tissues. Ultrasound is the only common imaging modality that does not have a nuclear basis. However it must be recognised that the separation of imaging based on modalities is artificial as all of these are but tools used either alone or in combination to arrive at the diagnosis and plan treatment in the most cost-effective and efficient way. In addition it is inescapable that the appropriateness of modalities would depend on the availability of equipment and expertise such that what is being practised by the most advanced nations may not be possible let alone expected in a less developed nation. The phenomenal development of computer processing has led to revolutions in imaging, for example the ability to do 3-D reconstructions of data-sets allowing for techniques such as virtual endoscopy for the assessment of the colon, bronchi or even blood vessels. In recent years, over-laying and fusing images from different modalities has provided additional medical information to improve diagnosis and to localise and gauge the severity of disease. Other computer-based advances include computer-aided detection and diagnosis for breast and lung nodules, image management systems and radiological information systems. The development of digital imaging has prompted the development of teleradiology, the transmission of images electronically across wide distances, which can provide remote areas rapid access to specialists for the interpretation of diagnostic imaging procedures.

And now we are entering a new age of Molecular Imaging, the ramifications of which are not easy to predict but will certainly be extensive. This technique involves the use of specific tumour or gene markers to facilitate disease detection and to determine treatment response at the cellular or gene level much earlier than would be possible with current imaging modalities. The traditional role of diagnostic imaging, as its name implies, has been in the diagnosis of infectious disease such as tuberculosis and it remains an essential tool for use in the setting of fractures. However, image-guided interventional radiology is rapidly developing as a minimally invasive and cost-effective modality of treatment. In this technique, medical imaging (ultrasound, fluoroscopy, CT and even MRI) is used to guide the radiologist in therapeutic manoeuvres such as angioplasty, embolisation, radio-frequency and ethanol tumour ablation, local chemotherapy delivery and abscess drainage, often most of these under local anaesthesia and sedation only. These techniques are less invasive than traditional surgical techniques, allowing the patient to recover much faster, and they are also less expensive. Although diagnostic imaging technology has made remarkable advances in the last few years, these advances have come with a price, and that price is not only financial. The more advanced imaging equipment also requires a much more advanced support structure. Therefore, the needs of Member States cannot be solved simply by the provision of equipment alone. Technology transfer requires an appropriate level of physical infrastructure (adequate and stable power supply as well as controlled cooling and humidity and dust control) and technical expertise to enable the equipment to function properly. Another important area is that of quality assurance and quality control, both essential for ensuring reliability. It also requires the training and development of local medical expertise since physicians are responsible for the diagnosis and treatment of patients and equipment is simply the means to achieve the desired end.
Proposed Programme Strategy Due to the vital importance of imaging in both the diagnosis and management of disease processes, there is a need for a greater coherent international effort to help the developing nations create strategies for the incorporation of imaging into their healthcare systems. To meet the needs of such countries, a comprehensive programme is required to take into consideration the availability of local expertise (medical and technical), the infrastructure (stable electrical supply, water and air-conditioning) and the disease pattern or burden. In short, the total solution requires coordinating Agency efforts with those of other partners. The Agency already has programmes in nuclear medicine and radiation therapy supported by activities in dosimetry and medical physics. Through the TC fund the Agency already supports projects in some areas of diagnostic imaging, dosimetry and radiation protection, but it lacks a comprehensive programme to provide a systematic approach focusing on the medical aspects of imaging science and including all the imaging technologies (Appendix 1). Member States require the Agencys assistance in this area since no single UN organisation has the resources or the expertise to do the entire job without help. In considering the role the Agency could play in supporting the development of effective diagnostic imaging in developing countries the meeting recognized that there are, broadly speaking, two problems both of which are on a daunting scale:

Scenario A The basic level: The need for the provision of the most basic of medical equipment and services to those essentially under developed nations who have virtually no infrastructure or trained personnel. Scenario B The advanced level: The need to improve and upgrade the established medical equipment and medical services in those developing countries who have them and who have some infrastructure and trained work force to support them. It should be recognised, however, that in some countries elements of both problems co-exist in different locations. The Agencys approach to these two different scenarios would have to be somewhat different, and these approaches will be discussed in more detail shortly. However, there are certain general principles which should underlie any comprehensive program in diagnostic imaging, and these should be considered first: i. Sustainability for the safe and effective use of imaging technology requires a long-term commitment by the Member State to ensure the necessary physical and technical infrastructure as well as on-going professional development of existing and newly recruited staff. Indeed, as shown in its existing programmes, by providing expertise and independent advice during project formulation, equipment specification and purchasing, facility commissioning and training of medical and support staff, the Agency is able to produce significant impact on the health care agenda of Member States. ii. Appropriate levels of technology need to be identified. Careful consideration must be given to the type of imaging equipment best suited to the purpose in different centres and situations. There are now a number of diagnostic imaging modalities and any comprehensive plan for diagnostic imaging must include all these modalities because they each have their own roles and advantages and disadvantages. Basic radiographic units and ultrasound equipment (particularly portable units) are the least expensive, require the least infrastructure and can be operated by personnel with only basic training. These modalities can be most widely disseminated and they provide excellent diagnostic support for primary medical care. Secondary referral centres that have surgeons and other medical specialists or easy access to such support could benefit from fluoroscopy, a basic CT, more sophisticated ultrasound as well as radiography. However, these modalities require a more stable infrastructure and more highly trained personnel to function effectively. The most sophisticated imaging equipment, MRI, high-end CT, nuclear medicine and PET is most logically placed in tertiary referral centres because it requires the most extensive and reliable infrastructure and personnel with the most advanced training. iii. As a corollary to this, balance must exist between the technology levels in diagnosis and treatment. For example, there would be little gain in providing advanced diagnostic capabilities for patients who have cancer or heart diseases to a centre that has no relevant associated local or regional therapy capabilities, or vice-versa. There is a need to define the medical needs and present capabilities of any country or centre requesting assistance under this programme. Agency tools such as the Country Programme Framework could play a key role in this process.

iv. Structures for continuing assessment and validation must be an essential part of any project, and any projects supported should be sustained for up to at least 10 years with the ultimate goal of handing over responsibility to local control when appropriate. There are also certain practical considerations that would be important in the development of a program in diagnostic imaging: a. The Agency should work with existing programmes and initiatives whether its own, whether local, or whether involving other governmental, inter-governmental or nongovernmental agencies. It should be a facilitator in these matters. In particular, in the matter of basic imaging in the less developed world it should seek to collaborate with the WHO, PAHO and ISR. b. Where training is the issue, the Agency should seek to tap into local facilities and programmes where these exist or into the existing courses of bodies such as the RSNA, EAR, ISR, IOMP and the equipment manufacturers. Setting up of new tailored programmes in collaboration with such bodies should be the aim where no pre-existing programme is appropriate to needs. Training should be appropriately practical and hands-on where possible and should match available local resources. Where some level of local expertise exists the aim should be to use this as a resource to train more widely in the region. This will help achieve diffusion of expertise. At the same time, the level of expertise of already trained individuals should be appropriately raised by organising training, elsewhere, regionally or internationally. The approach to the two different scenarios described above will now be discussed in more detail. Scenario A. Basic Level In areas where there are only very basic health services available, the provision of basic diagnostic imaging equipment can be of great value. This would include a simple x-ray machine and a basic ultrasound unit. This type of equipment is generally quite reliable and requires only limited physical resources such as an electrical supply. It does not necessarily require trained technologists, because other medical personnel can be trained to use it. It also does not necessarily require the presence of radiologists, because other physicians can be trained to provide preliminary interpretation of the images. The hard copy images can be sent to a more advanced regional center where radiologists are available for final interpretation. If appropriate facilities are available, teleradiology might also be used. Clearly the provision of this type of low technology equipment, with appropriate training, should be the goal in the least developed regions. Currently, the WHO, PAHO and ISR are providing basic equipment and training in a handful of centres in the under-developed world to create local centres of excellence in low-technology imaging. Representatives from some of these organizations who were at the meeting indicated that they would welcome collaboration with the Agency. The Agency should approach all these organizations to determine how it could best support their existing programs, perhaps by enabling more personnel from the least developed areas to attend the existing training programs of these organizations. Scenario B Advanced Level

The Agency can play a more pro-active role in supporting the development of advanced diagnostic imaging in developing countries, because it has already considerable relevant experience through its nuclear medicine and radiation therapy programs. The problems associated with Scenario B are already being tackled in regard to radiation therapy equipment by the IAEA so there is a track record in the Agency in this area. It would clearly be appropriate for the Agency to add a Diagnostic Imaging dimension to its activities since therapy and diagnosis are inextricably linked. It also follows that some concentration of efforts in the diagnostic imaging field should be in those regions already supported in therapy. Here the emphasis will be more on advanced technology such as CT, nuclear medicine and MRI. PAHO is also active in this area and collaboration with PAHO would be fruitful. There are a large number of national hospitals, which have at least one of the more advanced modalities for diagnostic radiology in the developing countries. Thus, the number of staff involved in diagnostic radiology is large in each country. Accordingly, it is very difficult to meet the national needs for training using the traditional approach of the IAEA of sending individual fellows abroad for training. To achieve this goal in a cost effective manner, it is necessary to emphasize a train the trainers approach to maximise the impact of training programs. In order to ensure the effectiveness of this approach in a given country, it is necessary to identify an appropriate collaborative centre to act as a focal point at the national level and to be considered as a reference centre in the country. This will involve a great deal of responsibility in such institutions, as they have to coordinate and carry out all the activities. To maximize the impact at the national level, the Agency should serve as coordinator/facilitator to enhance the co-ordination between national institutions and governments. This coordination may be formalised by encouraging governments to develop a network of participating radiodiagnosis departments, and to assign formally the national focal point institute the responsibility for ensuring proper linkages at the country level. In order to enhance TCDC, it is possible to identify certain regional resource units/ collaborative centres to help in conducting training and to provide advice to other developing countries. This is in response to TC Strategy in which the Agency intends to strengthen regional cooperation by encouraging the more advanced national institutes within regions to contribute fully to solving problems within the region. This type of cooperation will ensure the most effective use of the limited financial resources under the Agencys TC Programme. International centres of excellence could also play a role in providing advanced training. The IAEA, Board of Governors has endorsed the TC Strategy, which, among other elements, features the concept of expanding the impact outputs and outcomes through partnerships with other organizations in the field of activity such as governmental, professional, intergovernmental and non-governmental international organizations. The Agency should use the existing courses of bodies such as the professional societies, namely ISR, RSNA, EAR, CIR, AOSR, WFUMB, WFNM and continental imaging societies AOSR, ANZCR, CIR, EAR, IOMP for training radiologists, ISRRT for training radiographers and medical technologists and IOMP and EFOMP international training programmes for training medical physicists. For example, in its radiation oncology and medical physics programs, the Agencys recent cooperation with ESTRO in conducting training for East European countries, has demonstrated the effectiveness of such collaborative training programmes. There should also be collaboration in this area using the existing training programmes of such international bodies as WHO and

PAHO. Equipment manufacturers should also be invited to participate, in providing specialised training programmes for QA and maintenance of medical imaging equipment. Training projects are best delivered at the point of need even if this involves foreign faculty. There should be a strong practical (hands-on) component to the education and students should be recruited regionally to ensure dissemination of skills within the area of need. Resource material should be provided for project participants for prior study. To achieve sustainability and self-reliance, those involved should be encouraged to recognize that education is a lifelong process requiring continuing professional development. Training could be conducted through a variety of structures such as: 1. National workshops with the help of the Agencys experts in collaboration with the national focal points centres. Regional workshops/training courses using the existing training programmes/curricula for trainers. Individual fellowship/group fellowships at international collaborative centres/centres of excellence.
In developing its projects IAEA should, as a facilitator, consider the following: A needs assessment in respect of both educational and local human and technical resources. Benchmarking of staffing and equipment levels should be used in priority setting. Determination of the existence of available educational programmes by region and by nation.
The following types of projects should then be considered: Educational projects (for all relevant professional groups) Implementation of standards (e.g. acceptance and quality control testing) Implementation of guidelines (e.g. referral criteria, screening) Upgrade of existing imaging equipment to support advanced radiation treatment and educational initiatives in conjunction with such upgrades. On those occasions when entirely new projects are developed there may be little existing infrastructure and the project implementation will tend to occur in the longer term.
The projects will be of varying length and complexity. The projects should be directed to any or all of the following, recognizing the availability of local resources: Medical physicists
Radiologists Radiological nurses Allied scientists and technologists Other health care workers with different levels of expertise. Identification of relevant professional groups is a pre-requisite for implementation. For these programmes to succeed national administrations must recognise and validate qualifications that are primarily academically recognized. National authorities should be encouraged to legislate recognition of graduates, e.g. through health professions acts and certification. Such recognition should ultimately meet international standards when they are formulated.

The role of IAEA will include aspects of funding. Matching funding from local resources, IAEA and other organisations in differing combinations should be sought. IAEA should continue to be involved in funding international study for students and teachers. Governments may participate in these IAEA programs acting on their own initiative or in response to needs identified by organizations in their own countries, such as universities, or other national agencies. The IAEA should advertise its interest in supporting bids for participation in the above projects and in such bids: There should be an evidentiary basis for initiatives (appropriateness) Initiatives should be matched with available expertise and infrastructure Funding should have local matching component There should be well-defined and achievable objectives There should be a built in evaluation component involving examination of process and outcomes in both short- and long-term time frames.
IAEA should use its experience in quality assurance and process control to ensure continuous programme improvement. Moreover, IAEA should consider using external consultants to evaluate the initial bid and the projects progress and make appropriate recommendations as it evolves.
Conclusions The meeting agreed that the IAEA has proven capability for assisting the developing and lesser-developed countries through its human health division via the technical co-operation agreements with Member States. The Agency has put in place nuclear imaging and radiation oncology systems, which are appropriate for the needs of the countries taking into consideration the local situation. It has also managed to facilitate training for the human resources necessary to have such facilities function effectively.
The meeting envisioned a role for the IAEA in assisting the development of diagnostic imaging facilities in countries with the most basic needs. This would be done in cooperation with organizations like WHO, PAHO and ISR, which are already active in these areas. There is also a role for IAEA in the development of more advanced imaging modalities so that all Member States can reap the full benefits of what imaging has to offer in improving the level of healthcare available to citizens as stated in the strategic goal for technical co-operation with Member States that it shall increasingly promote tangible socio-economic impact by contributing directly in a cost-effective manner to the achievement of the major sustainable development priorities of each country. Action Plan The meeting defined the following specific activities that should be carried out by the Agency in the development of a thematic programme in diagnostic imaging: 1. The Agency will need in-house expertise and external consultancy arrangements and the individuals involved will have to establish their bona fides with all the collaborating groups. The Agency should, therefore, ascertain in-house competence, both radiological and technical, and where necessary seek appropriate external consultancy arrangements to work on this programme, which merits strong and expert direction. 2. The Agency should approach WHO, PAHO and ISR to discuss the possibility of collaborating with their programmes designed to assist developing countries in establishing basic diagnostic imaging facilities in the areas of greatest need. This activity could be developed very quickly, because these other agencies already have well-established programs in this area in place. 3. The Agency should develop a document defining the appropriate specifications for imaging requirements for centres of basic, secondary and advanced imaging. This document should also define the infrastructure, such as guaranteed power supplies and air conditioning, and technical and human resources, including technologists, radiologists and medical physicists, that are necessary for the equipment to function effectively and reliably on a long term basis. Equipment manufacturers could be of assistance in this process in providing guidelines for technical and infra-structural prerequisites for the suitable installation of their equipment. This activity is considered to be very important and should be started as soon as possible. It should be a short-term project. 4. The Agency should explore the feasibility of establishing a database of equipment, particularly advanced equipment in the developing countries. It was suggested at the meeting that equipment companies might be willing to cooperate in the development of such a database. There is some anecdotal evidence that non-UN initiatives to supply diagnostic equipment to the developing world may be in danger of being wasted as insufficient attention has been given to the principle of sustainability. Such a database might identify these situations, and Member States should be encouraged to consult the document described in the preceding recommendation prior to purchasing or acquiring new diagnostic imaging equipment. This action could be initiated in the short-term, but it would probably take some time to complete. 5. The Agency should undertake a needs assessment to determine the training requirements to support the development of more advanced diagnostic imaging facilities in developing

Tuesday, 27 May 09.00-10.30 Session 3a : Opportunities and Constraints Professional Societies Brian Lentle RSNA Holger Pettersson (Gerard D. Hurley) EAR Jan Labuscagne (Hans Ringertz) ISR
Industrys Viewpoint Pierre Renard Siemens Dietmar Gruidl GE
Donors Viewpoint Mahmoud Khene OPEC Fund

10.30-11.00 11.00-12.30

Break Session 3b : Opportunities and Constraints Technical Issues Martin Reed Canada Robert Nowotny - Austria Jos Carlos Da Cruz Brazil
Lunch Break Session 4a: Visioning - Defining the Future The session seeks to apply knowledge gained from the foregoing sessions to visualize a desirable future situation and address the question, Where do we want to go? Discussant: Peter Dawson
Break Session 4b: Convergence By characterising the necessary technical, institutional, operational and
partnership factors, participants will identify processes/steps that are required to make the link between the situation analysis and the future (vision) by providing answers to How? When? Where? With Whom? Defining the IAEAs role 19.00 Dinner

Wednesday 28 May

09.00-10.30
Session 5a: Summary and definition of working groups Chairpersons Summary of Previous Day Formation of Working Groups: the roles for the IAEA, Member States and Partners (Industry, donors and NGOs)

10.30-11.00 11.00-13.00

Break Session 5b: Defining A Future IAEA Programme on Diagnostic Radiology The starting premise of this session is that there is a problem which can be addressed using diagnostic radiology, a role, which although strategic and longer term in nature, may require some immediate action. Notwithstanding, the primary focus of IAEAs programme should be to address the technical and management requirements of national programmes. Key programme elements: Major Issues Partnerships - roles and responsibilities Institutional/Resource requirements Next steps/Follow-up: immediate, medium and longer term Pilot/Feasibility activities
13.00-14.30 14.30-15.30 15.30-16.00 16.00-18.30 Thursday 29 May 09.00-10-30
Lunch Break Session 6a: Working Group Discussions (cont.): Break Session 6b: Working Group Presentations

Session 7a: Planning Plan formulation and discussions

10.30-11.00 10.30-12.30

Session 7b: Drafting Groups Working Group Drafting
Lunch Break Session 8a: Concluding Working Group Drafting

12.30-13.30 13.30-15.30

15.30-16.00
16.00-18.00 Friday, 30 May 09.00-10.30
Session 8b: Conclusions and Recommendations
Session 9a: Reporting Review of Draft Report
10.30-11.30 11.30-12.30 12.30-13.00 14.00
Break Session 9b: Agreement on Chairpersons Report Closing by Ms. Ana-Maria Cetto, Deputy-Director General, TC Follow-up discussions with IAEA staff (NAHU/TCPA/TCPB)
Summary Information on Each of the Sessions
Session 1 Setting the Problem Context for Diagnostic Radiology within Health Care This session is intended to set the context of the problem of health care in under-developed countries with emphasis on the needs in diagnostic radiology. It will focus on the understanding of human, technical and financial resources as well as the general role of donor organisations.
Session 2 Present Status This session focuses on existing activities within the IAEA to understand some of the details of what is being done within the Health Care sector already. Lessons learned and methodologies used within existing programmes may help to guide the discussion on programme development and structure to be used in diagnostic radiology. Country Cases are meant to summarise experience and needs in the area of diagnostic radiology within Member States, essentially specifying their current situation and outlining what they foresee in the future.
Session 3 Opportunities and Constraints This session focuses on technical aspects (opportunities and constraints) on the use of diagnostic radiology. The presenters will also propose new ideas and identify future trends. The potential role for professional societies, industry and donor organisations will be discussed.
Session 4 Visioning The fourth session seeks to apply knowledge gained from the foregoing sessions to visualize a desirable future situation where national authorities and partners possess the technical know-how and human resources capabilities to effectively use diagnostic radiology in the diagnosis and precise status of human diseases. By characterising the necessary technical, institutional, operational and partnership factors, participants will in this session identify processes/steps that are required to make the link between the situation analysis and the future (vision). Some key issues to be answered include: What needs to be done to achieve national recognition of the problem (desirable future)? Where and how will diagnostic radiology contribute the most? What implementation strategies should be considered by the IAEA, and with what focus? The principal output of this session is conceptual agreement on questions of how, when, where and with whom relative to a possible IAEA programme on diagnostic radiology. More specifically: immediate, medium and long-term objectives and programme priorities; identification of roles and responsibilities; targets and timeframes; identification of actions that eventually converge at the TC project level. We will endeavour to move the developing concepts from the abstract to a concrete programme strategy. The Rapporteur plays a key role in recording the logical development of the discussion and any agreements reached, as they become the centrepiece of the report.

Sessions 5 & 6 - Summary and Formation of Working Groups Defining A Future IAEA Programme in Diagnostic Radiology This session in working groups is intended, to define a programme development strategy taking into account the major players. The starting premise is that the meeting thus far has concluded favourably that there is a problem where diagnostic radiology has a clear, well-defined and practical role to play in
managing malignant and other associated diseases. In this Session, the link to Session 3 comes from the conclusion that some form of programme development is feasible and the areas where the Agency can contribute are identifiable. Session 7 Planning This session summarises the presentation of the working group and prepares the outline of the necessary action to develop the Agencys programme in diagnostic radiology. The working groups reconvene to draft specific language for the conclusions and recommendations that will indicate clearly the next steps to be taken by all stakeholders.
Session 8 Concluding As a set of conclusions and recommendations for further action, it may be necessary to agree upon a list of prerequisites and enabling conditions required to establish an Agency programme in diagnostic radiology.
Session 9 Reporting The draft report has to be finished and agreed upon prior to closing the meeting.
APPENDIX 4 THEMATIC PLANNING MEETING ON DIAGNOSTIC RADIOLOGY VIENNA, AUSTRIA MAY 2003 List of Participants
Dr. Basri J. J. ABDULLAH Dept. of Radiology Faculty of Medicine University of Malaya Lembah Pantai Kuala Lumpur 59100 MALAYSIA Tel.: Fax: Email: basrij@um.edu.my Dr. Caridad BORRAS 1501 44th Street N.W. Washington, DC 20007 UNITED STATES Tel.: Fax: Email: borrasc@hotmail.com Dr. Ricardo Renzo BRENTANI Hospital do Cancer Rua Professor Antonio Prudente 109 01509-010 Sao Paulo BRAZIL Tel.: Fax: Email: rbrentani@ludwig.org.br Dr. Jose Carlos DA CRUZ Chief Physicist Hospital Israelita Albert Einstein Avenida Albert Einstein 627/701 05651-901 Sao Paulo, S.P. BRAZIL Tel.: 0486 Fax: 1412 Email:josecarlosc@einstein.br Dr. Peter DAWSON
Dr. Jan H. LABUSCAGNE International Society of Radiology, Radiology Society of South Africa P.O. Box 3475 Cresta 2118 SOUTH AFRICA Tel.: Fax: Email: janlab@labven.co.za Dr. Brian C. LENTLE 7997 Turgoose Terrace Saanichton BC V8M 1V4 CANADA Tel.: Fax: Email:blentle@shaw.ca Dr. Harald OSTENSEN World Health Organization Avenue Appia 20 CH-1211 Geneva 27 SWITZERLAND Tel.: 3649 Fax: 4836 Email:ostensenh@who.int Dr. Martin REED Children's Hospital Health Sciences Centre 840 Sherbrook Street Winnipeg R3A 1R9 CANADA Tel.: 2603 Fax: 1402 Email: mreed@exchange.hsc.mb.ca Dr. Holger PETTERSSON
International Society of Radiology 18 Green Lane Chesham Bois Amersham, Bucks HP6 5LQ UNITED KINGDOM Tel.: Fax: Email:phda728222@aol.com Dr. Gerard D. HURLEY Dept. of Radiology Adelaide and Meath Hospital Tallaght Dublin 24 IRELAND Tel.: Fax: Email: gerard.hurley@amnch.ie Dr. Boudjema MANSOURI Centre Hospitalo-Universitaire de Bad El-Oued Alger 16000 ALGERIA Tel.: Fax: Email: radiobeo@ibnsina.ands.dz

Mr. Royal F. Kastens Head, Concepts and Planning Section Division of Planning and Co-ordination Department of Technical Co-operation Email: R.F.Kastens@iaea.org
Ms. C. Nelima Okhoya Programme Planning Officer Concepts and Planning Section Division of Planning and Co-ordination Department of Technical Co-operation Email:C.N.Okhoya@iaea.org

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Downloaded from cancerres.aacrjournals.org on June 8, 2011 Copyright 1998 American Association for Cancer Research
[CANCER RESEARCH 58, 1850-1859.

May 1. 1998]

Somatostatin Receptor Subtype Specificity and in Vivo Binding of a Novel Tumor Tracer, 99mTc-P8291
Irene Virgolini,2 Maria Leimer, Hirsch Handmaker, Secondo Lastoria, Claudia Bischof, Pietro Muto, Thomas Pangerl, Doris Gludovacz, Markus Peck-Radosavljevic, John Lister-James, Gerhard Hamilton, Klaus Kaserer, Peter Valent, and Richard Dean
Departments of Nuclear Medicine /I. V. M. L. C. B. T. P. D. G., Gastroenterologe M.P-R. Surgery G.H. Pathology K.K., and Internal Medicine I. Division of Hematolog\ [P. V.}, University of Vienna, A-1090 Vienna, Austria; Arizona Institute of Nuclear Medicine, Phoenix. Arizona 85016 [H. H.J; Department of Nuclear Medicine, National Cancer Institute, 80131 Naples, Italy S. , P. M.]: and Diatide, Inc. Londonderry, New Hampshire 03053 J. -J., R. D. L L

ABSTRACT

Recent data suggest that somatostatin receptors (SSTRs) are expressed on various tumor cells. High-level expression of SSTR on the tumor cell surface provides the basis for the successful clinical use of radiolabeled ligands for the in vivo localization of tumor sites. We have characterized the in vitro binding properties of the novel SSTR ligand "mTc-P829 using primary human tumors (carcinoids, breast cancers, intestinal adenocarcinomas, pheochromocytomas, small cell and non-small cell lung cancer, and melanomas; n = 28), various tumor cell lines, and COS7 cells transfected with the human SSTR (hSSTR) subtypes 1, 2, 3, 4, and 5. """TcP829 bound to primary tumor cells and tumor cell lines with high affinity and high capacity. The dissociation constants (A,,I ranged between 1 and 20 IIM. '"""Tc-1'829 also bound with high affinity to the transfected hSSTR2 (Ka, 2.5 nM), hSSTRS (Ad, 2 nM), and hSSTR3 (K,1.5 nM). Binding of "'""Tt-PH29 to hSSTR3 was found to be displaceable by unlabeled P829/([ReO]-P829), SST-14, and vasoactive intestinal peptide (VIP; IC50, 2 nM) and, less effectively, by Tyr'-octreotide (IC50, 20 nM). In contrast, the binding of 99mTc-P829 to hSSTR2 and hSSTRS could be displaced by P829/([ReO]-P829) and Tyr'-octreotide but not by VIP. 99l"Tc-P829 scintigraphy revealed in vivo binding to primary or metastatic tumor sites in seven of eight patients with breast cancer and six of six patients with melanoma. In summary, our data show that 99n>Tc-P829 binds with high affinity to many different types of primary and cloned tumor cells. Furthermore, our data identify hSS TK2. the VIP acceptor hSSTR3, and hSSTRS as the respective target receptors. Because these receptors are frequently expressed at high levels on primary tumor cells, 99mTc-P829 appears to be a promising novel peptide tracer for tumor imaging.
strated (1, 2, 5). In fact, such tumors frequently coexpress VIP and SST/OCT binding sites. An interesting phenomenon is that VIP and OCT can cross-compete for binding to tumor cell membrane receptors (2). The molecular basis of this phenomenon could not readily be explained thus far. However, the molecular cloning of SSTR and VIPR has recently provided new insights into the biology and interactions of VIP and SST. To date, five different human SSTRs (6-13) and two different VIPRs (14-19) have been characterized in detail and have been cloned. Using trans fected peptide receptors, hSSTRS has recently been identified as a potential common acceptor site for both SST/OCT and VIP (20). Several efforts have been undertaken to identify hSSTR subtypes expressed in primary human cancers (for a review, see Ref. 21). A number of observations suggest that hSSTR2 is expressed in many different tumors, including neuroendocrine tumors and breast cancer (22). However, other hSSTR subtypes have also been detected (23, 24). We recently have demonstrated expression of hSSTRS mRNA in a variety of human tumors, including breast cancers, melanomas, and neuroendocrine tumors (21, 25). Because this receptor (hSSTR3) was found to bind both VIP and OCT (20), it was hypothesized that this site is responsible for the observed cross-competition of these peptides in primary human tumors. Despite the clinical usefulness of "'In-OCT (3) and 123I-VIP (4), several attempts have been made to label hSSTR ligands with 99mTc because of its optimal decay properties and cost effectiveness (2628). Recently, P829, a peptide containing a sequence that mimics the binding domain of SST, has been identified as a suitable hSSTR ligand that can be labeled with 99mTc (29). However, the spectrum of human tumors that can be visualized by 99mTc-P829 has not been defined yet. Also, the binding behavior of this novel hSSTR ligand onto various subtypes of hSSTR is not known. The aims of the present study were to evaluate the binding characteristics and hSSTR subtype specificity of 99mTc-P829, as well as the binding affinity of this novel tracer for primary human tumors.

INTRODUCTION The high-level expression of peptide receptors on various tumor cells as compared with normal tissue or blood (1,2) provides the molecular basis for the successful use of radiolabeled SST* analogues (such as OCT) and VIP as tumor tracers in nuclear medicine (3, 4). Thus, using primary tumor cells or cell lines, specific binding of both VIP and OCT to the cell surface of various tumors has been demonReceived 9/19/97; accepted 3/3/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' These studies have been supported in part by the Austrian National Bank (Jubilums fonds Projects 5460 and 6512) and by a Foundation of the Mayor of the City of Vienna. Parts of the studies were sponsored by Diatide. Inc. 2 To whom requests for reprints should be addressed, at Department of Nuclear Medicine. University of Vienna, Whringer Grtel18-20. A-1090 Vienna. Austria. Phone/Fax: 43-1-40400-7835; E-mail: irene.virgolini@akh-wien.ac.at. 3 The abbreviations used are: SST, somatostatin; SSTR, SST receptor; hSSTR. human SSTR; VIP, vasoactive intestinal peptide; VIPR, VIP receptor; 123I-VIP, '"l-labeled VIP; IC50, concentration of unlabeled ligand necessary to induce 50% inhibition of labeled ligand binding; Kd, dissociation constant (concentration of labeled ligand necessary to produce half-maximal binding); LNN, lymph node mtastases;NSCLC, non-small cell lung cancer; OCT, octreotide; "'In-OCT. '"In-labeled OCT: P829. novel SST-14 ana logue; |ReO]-P829, oxorhenium complex of P829; PMNC, peripheral blood mononuclear cell; SPECT, single-photon emission computed tomography; SSM, superficial spreading melanoma.

MATERIALS

AND METHODS
Synthesis and Labeling of P829. The peptide P829 was synthesized using solid-phase peptide synthesis techniques and /v'-(9-fluorenyl)methoxycarbonyl chemistry and was purified by preparative high-performance liquid chromatography as described (29). [ReO]-P829 was prepared by ligand exchange using tetrabutyl ammonium oxorhenium tetrabromide, which was prepared according to Cotton and Lippard (30). For practical use, we formulated an instant kit containing 50 /ig of the P829 peptide. This P829 kit was reconsti tuted with 500 MBq-3 GBq "'"Tc-pertechnetate (CIS Bio-International, Paris, France) in a final volume of 1-5 ml. The reconstituted product was heated for 15 min and then kept at room temperature for 45 min. An aliquot of this preparation was used for the in vitro series of experiments. The radiochemical purity of the 99mTc-P829 product was determined by instant TLC (ITLC-SG; Gelman Sciences, Ann Arbor, MI) developed in saturated saline [99mTc-P829 and 99mTc-microcolloid, relative fraction (rf) 0-0.75] and ITLC-SG developed in pyridine:acetic acid:water (5:3:1.5) for determination of "Tc-microcolloid

BINDING

PROPERTIES

OF ''"Tc-PK29

Table 1 Binding if v'"Tc-PH29. 9 Approximately 500 MBq WmTc-P829 were injected i.V. and planar and SPECT images were acquired within 24 h after injection. 99mTc-P829 identified primary tumors and mtastasesas indicated in the Table. NP, not performed. Detection of tumor lesions by scintigraphy with Patient No./initials Site of disease" WmTc-P829 "in-OCT I-VIP
Breast cancerI/EE2/SB3/RS4/JA5/HB6/BW''7/LT*8/CCfr9/SM'IO/NE'1 (right)Primary cancer breast (right)Primary cancer breast (left)NHLbreast cancer (left)Primary (MALToma) of the breast (left)Supraclavicularian cancer ductal breast (left)Multiple LNN positive>l() positiveNegative1/1 tissueRecurrent in bone, liver, soft LNN (left)LNN. lobular breast cancer thoraxPrimary left lower positiveNPNPNPNPPositivePositive1/1 tumorPrimary ductal tumorPrimary lobular I/RE'12/TS''Melanomas1/LR'2/CF3/SA*4/VS"-'5/MR'"6/BMC'"''7/TJc8/KL''-'CarcinoidsI/RE*"2/TK"'rSCLCI/RM'NSCLC15/SP1" tumorPrimary ductal tumorPrimary ductal choroid)Primary(right SSM toe)LNN SSM (left groin)LNN (left axilla)LNN (left groin)LNN and right (left axilla)s.c. (right metastasiss.c. mtastasesLNN. axillaBone right mtastasesLNN, groins.c. left, right mtastasesLung positiveNPNPNPNPNPNPNP's metastasisPrimary NMs.c. mtastasesLNN mtastasesLiver metastasisLNN (abdomen)Primary mtastases tumorPrimary
positive1/1 positive>!(> positive1/1 positiveI/I positive3/3 positive1/1 positive5/5 positive4/4 positive2/2 positiveI/I
tumorPositivePositivePositivePositiveNegative1/1 melanoma:already lymphoma; MALToma. mucosa-associated lymphoid tissue tumor; NM. nodular non-Hodgkin' NHL, small cell lung cancer;NPNPNPNPNPNPNPNPNPNPPositiveNPPositiveNPN not performed. was' Primary tumor scintigraphy., resected at the lime of From these patientsPrimary specimens were obtained for in v/fm receptor analysis (seealso tissue Table 2).NPNPNPNPNPNPNPNPNPPositivePositivePositivePositivePositivePositivePositivePositivePositivePosi

(rf 0-0.25).

The radiochemical
purity of 99mTc-P829 was >90% in all exper
iments. Preparation of '"In-OCT. ' ' 'In-OCT was prepared by reacting 10 ig f o diethylenetriaminepentaacetic acid-OCT for 10 min at 22Cwith 100-200 MBq of '"In-Cl, according to the manufacturer's description (Mallinckrodt Medical. St. Louis. MO). This product was routinely applied for scintigraphy. and an aliquot of this preparation was used for in vitro studies. Preparation of Primary Tumor Cells. Written informed consent was obtained from all patients undergoing surgery. Tumor tissue specimens (0.5-1 ml) were collected at surgery and immediately placed into liquid nitrogen. The diagnoses were established by histological examination and immunohistochemistry according to WHO criteria. In particular, tissue was analyzed in patients with carcinoids (n = 2), pheochromocytomas (n = 4), colonie adenocarcinomas (n = 3), ductal breast cancer (n = 4), lung cancer (n = 2). and melanomas (n = 12). Tissue was stored at 70C until it was used for in vitro studies. Tumor cell membrane fractions were prepared according to established techniques (2). Briefly, tissue was thawed, cut into pieces, put into 50 mM Tris-HCI buffer (pH 7.5) and 5 mM MgCl-,. and homogenized by means of a glass homogenizer. The cell homogenate was centrifuged at 5000 x g for 10 min at 4C. washed, and resuspended in assay buffer containing 50 mM Tris-HCI (pH 7.5) and 5 mM MgCl, at a concentration of 100-1000 /xg protein/ml, and measured by the method of Bradford (31). Preparation of Tumor Cell Lines and Blood Cells. The epidermoid carcinoma cell line A431; the colonie adenocarcinoma cell line HT29; the melanoma cell line 518A2; and the breast cancer cell lines T47D. BT20. ZR75-1. and MCF7 were purchased from American Type Culture Collection (Rockville. MD). The basophil cell line KU812 was kindly provided by Dr. K. Kishi (Nijgata University, Nijgata. Japan), and the mast cell line HMC-1 by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN).

Cell lines were cultured in RPMI 1640 (A431. HT29, T47D. BT20, ZR75-1. MCF7. and KU8I2) or in Iscove's modified Dulbecco's medium (HMC-1) supplemented with 10% PCS, i.-glutamine. and antibiotics in 5% CO,/95% O2 air at 37C.Cells were fed two to four times per week. Adherent cells were passaged with trypsin (Worthington. Freehold, NJ) after confluency was reached. Before being used in binding experiments, cells were washed in 50 mM Tris-HCI buffer (pH 7.5) and then resuspended in 50 mM Tris-HCI assay buffer (4C;pH 7.5), containing 5 mM MgCl, and 0.1% BSA. About 5 X IO7 cells suspended in 5 ml were used for one series of experiments. Normal PMNCs and blood platelets were prepared according to published techniques (32). Transfection. Plasmids containing the receptor cDNAs for the human SST receptors (hSSTRl through hSSTR4) were kindly provided by Dr. G. Bell (Howard Hughes Medical Institute. Chicago, IL). The plasmid carrying hSSTR5 was kindly provided by Dr. A. M. O'Carroll (NIH, Bethesda, MD). Correctness of the receptor clones was checked by agarose gel analysis of restriction fragments. Furthermore, a complete sequence analysis was per formed for each clone. Isolation of plasmid DNA was carried out with the Qiagen plasmid purification kit (Qiagen. Hilden. Germany). COS7 cells were grown in RPMI 1640 containing 10% fetal bovine serum and antibiotics (Life Technologies, Inc., Vienna, Austria). Transient transfection of COS7 cells with different receptor cDNAs was performed using the yV-[l-(2.3-dioleoyloxyl)propyl]-A',A'./V-trimethylammoniunimethyl sulfate transfection reagent (Boehringer Mannheim) according to the manufacturer's description. Twenty-four h after transfection. the medium was exchanged, and after 48 h in culture, the cells were harvested, an aliquot was recovered for RNA extraction and Northern blot analysis, and the remaining cells were used immediately for binding studies.

OF "Tc-P829

Table 2 "'Tc-P829 binding to primary tumors in vitro and mRNA expression Tumor tissue was removed during surgery, and tumor membrane fraciions were prepared as described in the texl. All concentrations listed are in nM. The maxefers lo Ihe lolal r number of specific binding siles esiimaied from ihe binding of 99mTc-P829 in Ihe concenlralion range of [99mTc-P829] listed, and Ihe K value is the dissocialion conslanl for Ihis inleraclion. The IC50s indicale the concentralion of unlabeled ligand necessary lo reduce Ihe specific binding of 99mTc-P829 (al the concentration of [99n'Tc-P829] used in the assay) by half. NP. not performed. Northern blot score: + + +, very strong expression; + +, strong expression; +, weak expression; -, no expression. experimenlsPalient Saturalion values)["mTc[ReO]experimenls (IC50 Tyr1expression2++NPNP+NPNP-(+)NPNPNP++NP+

OCT<30 P829 P829]32.50.90.932.511.72.75.032.58.78.71.01.0582691.70.10.2NP10.8NP12.50.83551NP0.15P829/ 1NP3 No./inilialsCarcinoidsI/RF2/TKPheochromocylomas3/GO4/TO5/BJ6/TMColonieadenocarcinomas7/HW8/SK9/HADuclal SST-14 diagnosisLiver melasiasis"'Lymph melasi."'Normal node mucosaPrimary jejunal lumorPrimary lumorPrimary tumorPrimary tumorPrimary IO123.6 X IO121.2 X.0-2600.3-130.16-354.3-2720.9^ 10"1.5 X 10"3.0 X IO121.7 X 10"Negative1.8 X 3003 NP NegativeNegative 3 Negative30 Negative Negative3NP NP4 NP NP1.++ +NPNP+NPNP+ +NPNPNPNPNP+(+)NP+NP+
+Negalive NPNP NP35 NP3.3 NPNPNPNP
lumorPrimary lumorNormal mucosaPrimary colonie lumorPrimary cancer10/SMll/NE12/RE13/TSLung breast lumor"Primary lumor"'Primary lumor"Primary M)SCLC"NSCLC (Fig. lumor"
IO129 X NegativeIO NP IO12Negali X 10.9^111.6-711.6-713.6-2638.5-2690.3-130.9-410.01-19.41.7-75.40.18-71.5NP0.1-67NP'2.1-96.51.1-73(Fig. NPNegative NP NP3 Negative ve3 NP10 IO123 X NP3 NP NPNP IO121.8 X IO121.2 X 10"4.2 X IO119 X NP NP NP 500 NP 2 NPNP +NP500NP

++ + +NP+ +NP+ +

NP<60 NP<50 NP5 NP<3 cancer14/RM15/SPMelanomas16/SE17/TJ18/NM19/LR20/HH21/ZJ22/RH23/VS24/PL25/KL26/PX27/MR28/BMCPalienl IO12''6.2 X <11 IO121.2 X NPNP IB)Primary (Fig. (SSM)Primary lumor (NNM)"Primary lumor IO123 X IO12NP1 X

++NP+NPNPNPNP+

+NP++NPNPNPNP+ + 1C)NP(Fig. NPNPNP ID)NPNPNPNP+ +NPNPNPNP+NPNP+NP+ (Fig.
NP8 NP NPNegalive 814 NP NPNP 2.5NP NP (SSM)Primary lumor NPNP (SSM)''lymph lumor IO12NPNegative1 X NP1 NP NPNPNP +NPNP++NPNP+ + +NPNPNPNP+++NP melasiasislymph node NPNegative NP NP0.8 Negative NPNP melasiasislymph node IO12NPNegative3 X melastasislymph node Negative4NP NPNPNP melastasis''s.c. node 5B)0.05-630.02-60NP0.1-23NP(siles/mg)3.0 4B + +NP++ +NP+++NP5NP+NPNPNPNPNP++N + + 5Negative NP metastasis"'*s.c. Negative1 Negative NP0.4 IO12NP6 X metastasis"'s.c. -Negalive +++ Negative8.6 2.3 metastasis"'s.c. NP ++ + ++ NegativeNP Negative melasiasis''s.c. X IO12NP151Negative15411.5155Negative2510152.41.65152NP1NPNegative1NPNegative1NP4.5NPDisplacement +NP3+ +NP4++ + NPNP NP NPNegalive 63 NegativeVIP NPSSTR-mRNA melasiasisP8294.3-2720.17-100.17-104.3-2721 " In these patients, In-OCT scintigraphy visualized the tumor site in vivo prior to its surgical removal. b In these patients, 123I-VIP scintigraphy visualized the tumor site in vivo prior to its surgical removal. * In this patient, In-OCT was used as a radioligand. *' In these patients, 99mTc-P829 scintigraphy visualized the melanoma tissues (see also Fig. 4).

RNA Extraction and Northern Blot Analysis.

Snap-frozen tumor speci

Radioligand Binding Assay. To evaluate expression of P829-binding sites on tumor cells, saturation and displacement studies were performed. In prin ciple, the assay conditions were the same as those reported previously for other peptide ligands (2). All saturation studies were performed under steady-state conditions at 4C.In saturation experiments, the intact cells or the membrane fractions were incubated with increasing concentrations of "mTc-P829 (typi cally 0.01-100 nM. unless otherwise specified) in the absence (total binding) and the presence of the unlabeled peptide P829 ( 1 /AM,nonspecific binding). Alternatively, SST-14 or [ReO]-P829 was used to assess the nonspecific binding. In displacement experiments, the intact cells or the membrane frac tions were incubated at 4Cwith 99mTc-P829 in the absence (total binding, 0.1-10 nM if not otherwise indicated) and in the presence of increasing concentrations (0.001-1000 nM) of the unlabeled ligands P829. [ReO]-P829, Tyr'-OCT, SST-14, and VIP. After incubation of intact cells for 45 min, the reaction mixture was diluted 1:10 with assay buffer (4C)and rapidly centrifuged (5000 X g for 10 min at 4C)to separate membrane-bound from free ligand. The resulting pellet was
mens were put in liquid nitrogen and homogenized in Trizol Reagent (Life Technologies) using a glass homogenizer. Total RNA from the homogenate was extracted following the Trizol extraction protocol, a modification of the RNA extraction described by Chomczynski and Sacchi (33). RNA of transfected and control COS? cells was extracted using the same technique. Integ rity of RNA was confirmed by agarose gel electrophoresis and staining with ethidium bromide. Northern blot analysis of receptor subtype expression was carried out as described (34). Northern transfer of 10-20 (g f total RNA was o performed using S&S Nytran membrane (Schleicher & Schuell, Vienna, Aus tria) by capillary blotting overnight. RNA was fixed to the membranes by UV cross-linking. Specific probes for Northern hybridization were generated by restriction cutting of the plasmids carrying the probes with the appropriate restriction enzymes. After separation with an agarose gel. the probes were purified with the Qiaex gel purification kit (Qiagen) and labeled using the Redivue random prime labeling kit (Amersham, Buckinghamshire, United Kingdom) and [32P]dCTP (Amersham). Hybridization was carried out as described (33. 34). Briefly, the membranes were prehybridized at 42Cin a hybridization solution containing 50% formamide, 5X Denhardt's solution,

washed twice with buffer and counted in a gamma counter for 1 min. Filters were presoaked with 0.1% BSA. 5X SSC (sodium chloride and sodium citrate), 0.2% SDS, and 100 ig/ml Specific binding was determined as the difference between total and non salmon sperm DNA. After 4 h of hybridization, the labeled probe was added specific binding. Binding data were analyzed according to Scatchard (35). In selected binding experiments, ' " In-OCT was used as a ligand for comparative in fresh hybridization buffer, and hybridization was carried out overnight. Thereafter, blots were rinsed twice at room temperature with 2X SSC buffer binding studies. containing 0.1 % SDS and then twice at 42Cwith 0.2 x SSC buffer containing In Vivo Studies: Patients and Gamma Camera Imaging. The adminis tration of 99"'Tc-P829 to patients was approved by the local Institutional 0.1% SDS and exposed to an X-ray film (Hyperfilm; Amersham). 1852

OF '9mTc-P829

4 99mTc-P829 (nmol/L)

11Tc-P829(nmol/L)

18S 28S
COcoCMcoco.CcoO-co(0.C^J.coco.CIO(T COco-C

actin

Fig. I. Specific binding of "mTc-P829 to a primary human breast cancer (A; Table 2. patient 13| and a NSCLC (B: Table 2, patient 15) The tumors were obtained intraoperatively, and tumor membrane fractions were used for the binding studies. These were incubated with increasing concentrations of "mTc-P829. The specific binding (shown) was calculated by subtracting the amount of "mTc-P829 bound in the presence of an excess of unlabeled P829 or [ReO]-P829 (1000 nM; nonspecific binding) from that bound in its absence (total binding). Saturation curves and Scatchard analysis (A and B. insets) indicated a saturable number of specific binding sites for Wn)Tc-P829 for both tumors. A single high-affinity binding class was demonstrable for the breast tumor displaying a Bma^ of 4.2 X IO11 sites (i.e., 720 fmol)/mg protein and a K f 2.4 nw. Scatchard plots derived for the saturation data of o NSCLC identified two classes of specific high-affinity 99nlTc-P829 binding sites with a Bmaxl of 6 X 10" (i.e. 1 pmol) and a m2 5.4 X IO'2 (i.e., 8 pmol) sites/trig protein and of a Kd, of 0.25 and a K2 f 5 nM, respectively. mRNA analysis by Northern blotting identified in the breast tumor the presence of hSSTRJ. hSSTR2, hSSTR4. and hSSTRS (O and o in NSCLC, the presence of hSSTR2, hSSTR3, and hSSTR4 (D).
Review Board and was performed according to the Declaration of Helsinki. All patients gave written informed consent to participate in the study. Eight patients with breast tumors and six patients with melanomas were studied as part of Phase Ila/IIb clinical investigations to evaluate 99mTc-P829 binding to tumor tissues in vivo (Table 1). Four patients had ductal breast adenocarcinomas and one a non-Hodgkin's lymphoma (mucosa-associated lymphoid tissue type). In three patients, the primary breast tumor had already been resected at the time of scintigraphy. These three patients presented with LNN and/or bone and soft tissue mtastases.Among the six melanoma patients, two patients had a primary SSM, and in five of the six patients, multiple sites of mtastases were present at the time of scintigraphy (Table 1). Scintigraphy with "mTc-P829 (370-550 MBq) was performed within 1 month following conventional stag ing procedures (biopsy, computed tomography and/or magnetic resonance imaging, mammography, and bone scintigraphy). One group of the patients who had the "Tc-P829 scan was also imaged by "'in-OCT. In a group of patients in whom tumor tissue was analyzed for expression of "mTc-P829 binding sites, I23I-VIP and/or "'In-OCT scintigra phy were performed prior to surgery according to Refs. 3 and 4, whereas "TC-P829 scintigraphy was not available. A summary of patients who underwent scintigraphy is shown in Table 1. Whole-body and planar scanning, as well as SPECT studies, were performed within 24 h after injection of 99mTc-P829. Planar and SPECT images were acquired either with an Orbiter II (melanoma; Siemens, Erlangen, Germany) or

X-40 (breast cancer; SMV, Twinsbury, OH) gamma camera equipped with a high-resolution collimator. The pulse-height analyzer window centered around the "Tc peak (140 keV) with a window width of 20%.The planar images were acquired with a matrix resolution of 128 X 128 pixels, 1000 kilocounts for the whole body, 300 kilocounts for the head and extremities, and 500 kilocounts for the chest and abdomen. SPECT was performed at 2 h postinjection using a matrix of 128 X 128 pixels, 64 steps (30 s/step). and a 360otation. SPECT images were r reconstructed in conventional axial, sagittal, and coronal projections, using either a Shepp Logan Hanning reconstruction filter (Orbiter II) or a Butterworth filter (SMV-X-40). Scans were viewed by two independent nuclear medicine physi cians. Using standard nuclear medicine techniques, foci of increased labeled peptide accumulation were considered as true positive sites of disease when corroborated by computed tomography and/or magnetic resonance imaging find ings or confirmed by histology after surgical biopsy.
RESULTS In Vitro Binding of WmTc-P829 to Primary Human Tumors and Mtastases.A variety of primary tumors were examined for expres sion of 99mTc-P829 binding sites. For this purpose, tumor membrane preparations were used. The primary tumors, as well as their mtas tases, bound 99mTc-P829 in a saturable manner, indicating the pres-

OF '"'"TC-P829

Table 3 99"'Tc-PX29 binding to tumor cell lines und SSTR mRNA expression Intact tumor cells or normal peripheral blood cells were used for the experiments listed. All values are listed in niu. The mas umber of binding sites estimated from the specific n binding of WmTc-P829 determined in the concentration range of |''9l"Tc-P829] listed, and the K0 value is the dissociation constant for this interaction. The IC50s indicate the concentration of unlabeled ligand necessary to reduce the specific binding of "mTc-P829 (at the concentration of |'Wl"Tc-P829] used in the assay) by half. NP. not performed. All experiments were repeated three to six times. Northern blot score: studyBmlx , very strong expression: + +. strong expression: +. weak expression: no expression.
(IC5()s)V1-51-510-201-510-15Negative1-510-200.55-151-55-101-510-151-510-303-53-5""'TC-P8296.7-10.56.7-10.55-10.56.7-10.51.1-27.29.9-11.31.1-86.4-7.80.2-1 study (mRNA)VIP subtype expression
[ReO|-P829Negative10-151-15Negative3-3010-300.3-310-301-53-103-10SST-14Negative10-15NP'Negative3-301-10NP10-30NPNP10 (sites/cell)0.6-1 3NP 5(+) 10s0.3-1 X +10-15 (+) ++ T47DBT20ZR75-IMCF7HT29KU8I2HMCIA431518A2*PlateletsPMNCs"mTc-P8290.8-740.8-740.8-741 IO50.2-1 X +Negative (+ ) + +( IO62-5 X IO51 X +NP (+ ) +NP +) IO6Negative0.8-1.5 X.9-740.1-21980.6-6000.1-12741.6-72170.1-750.17-560.4-56Saturation NP3-30 NP NP NP( IO50.3-3 X +3-30 (+ ) ++ (+)NP +> IO61-10 X IO53-30 X NP10-30 NP NP NPNP IO60.5-1 X IO60.9-1.8 X NPNP NP NP NPNP IO70.6-1.2 X IO60.5-1. X NP1-10 NP NP NP+ IOX IO124.2 X +3-10 (+ ) (+ ) ++ +NP ++ IO133-6 X IO34-6 X NP1-10 NP NP NP( X IO3Displacement (+ ) +4 +) " nlaxnd K given for the two high-affinity binding classes, where two values appear. a are For the experiments ' NP. not performed. with 518A2 melanoma cells, membrane fractions were used, and the sites/mg protein are listed.

PROPERTIES OF "Tc-PS

2.5 -,

m o>

CM CO Q_

T1Tc-P829(nmol/L)
20 T1Tc-P829 (nmol/L) 6-1

99mTc-P829 (nmol/L)

(/> O

C/5 O 0

CO O 0

r-. co o o

99mTc-P829

(nmol/L)

Fig. 2. Specific binding of 99mTc-P829 to COS? cells transfected with hSSTRl (A), hSSTR2 (B), hSSTR3 (O. hSSTR4 (D). and hSSTRS (E). COS? cells were transiently transfected with hSSTR I through hSSTRS. Intact transfected cells were incubated with increasing concentrations of l)I)mTc-P829. The specific binding () as calculated by subtracting the amount w of "mTc-P829 bound in the presence of an excess of unlabeled P829 or [ReO]-P829 (1000 nM; nonspecific binding, T) from that bound in its absence (total binding. A). Saturation curves and Scatchard analysis (B, C, and E, inserta) indicated a saturable number of specific binding sites for 'w"Tc-P829 binding to hSSTR2. hSSTR3, and hSSTR5. However, no specific binding of 99mTc-P829 could be observed for hSSTRl and hSSTR4. as indicated by unsaturable total binding (Aj and nonspecific binding (T). The respective binding data are listed in Table 3, which shows a Kd value of 2.5 nM for hSSTR2 (2 fmol/106 cells; i.e. 1500 sites/cell, n = 6), 1.5 nM for hSSTR3 (6 fmol/106 cells; i.e. 3600 sites/cell), and 2 nM for hSSTRS (6 fmol/106 cells; i.e. 3600 sites/cell). F. mRNA analysis by Northern blotting for control (i.e., nontransfected COS? cells; bottom) and COS? cells transfccled with the respective hSSTR subtypes 1-5 (top) used for these studies.

OF 9g"Tc-P829

Table 4 Binding of WmTc-P829 to SSTR subtype expressed on COS? cells COS7 cells were transiently transfected with hSSTRl through hSSTRS (see also Figs. 3 and 4). Intact transfected cells were incubated either with Wr"Tc-P829 or ' "in-OCT (data in brackets). The results listed indicate the median (range) of at least six independent experiments. The maxefers to the total number of binding sites estimated from the specific r binding of |'WmTc-P829] determined in the concentration range of [9MlITc-P829] listed; and the Kd. is the dissociation constant of this interaction. The IC50s indicate the concentration of unlabeled ligand necessary to reduce the specific binding of WmTc-P829 (at the concentration of |yt mTc-P829] used in the assay) by half. All values are listed in nM.
study(sites/cell)NegativeNegative1.500 (IC50s)KJNegativeNegative2.5(0.25-5)1.5(0.1-2)1.5(1-10)15
SSTRlSSTR2SSTR3SSTR4SSTR5"mTc-P829['"in-OCT]0.09-58410.09-6.90.1-5709[0.01-13.80.01-20000[0.03-960.4-2598[0.04-960.15-37[0.007-96Saturation
(0.3-3)1 (0.3-1)1 (1,200-10.000)720(240-1.200)3.600(600-10,000)1.200(1.000-4.000)NegativeNegative3.600 (0.3-3)1 (0.3-3)1 (<l-3)20 (0.2-10)20 (1-5)10(5-50)NegativeNegative1 (3-30)10(5-50)NegativeNegative3.5 (0.3-10)10 (5-50)NegativeNegative2 (30-50)NegativeNegative1 (5-50)1NegativeNegative]>300Ne

1.200-6.000)6.200(600-12.000)Displacement < (0.1-10)0.5 (0.3-30)1 (0.3-30)1 (3-30)5(1-30)VIPNegativeNegative]>500>IOO (0.5-10)99mTc-P829["'in-OCT]0.7-1320.03-1.30.7-9.60.14-7.60.7-5.60.15-7.80.19-1100.2-5.72.2-4.40.1-5.7|ReO]-P829Nega (<l-30)SST-14orP829NegativeNegative1 (1-30)Tyr3-OCTNegativeNegative3(<l-3)1
DISCUSSION lines (Table 3). The observation that a variety of tumors express significantly 99nTc-P829 binding to HSSTR2, HSSTR3. and hSSTRS (transfrants) was displaced by unlabeled P829/[ReO]-P829, by Tyr'-OCT, and by higher amounts of SST/VIP binding sites as compared with normal SST-14, with IC5()sin the nanomolar range, as indicated in Table 4. No peripheral blood cells and tissues led to the clinical use of radiolabeled substantial difference was observed for the displacement of the specific SST/OCT analogues (3) as well as of VIP (4) for the in vivo local binding of "mTc-P829 and ' " In-OCT bound to HSSTR2 and hSSTR3 ization of tumors expressing peptide binding sites. In the present study, the by unlabeled peptides. The rank order of potency for both ligands at novel SST binding behavior and receptor subtype specificity of the analogue 99n'Tc-P829 has been evaluated. The results of hSSTR2 sites was SST-14 = P829 = Tyr'-OCT VIP. The rank order our study show that 99mTc-P829 binds to many different types of of potency for 99mTc-P829 binding to hSSTRS sites was SST14 > P829 > VIP > Tyr'-OCT, and that for "'In-OCT was SST- tumor cells, including breast cancers, intestinal adenocarcinomas, and 14 > P829 > Tyr'-OCT = VIP. The rank order of magnitude for both melanomas. Furthermore, our study identities hSSTR2, hSSTR3, and hSSTRS as "'"Tc-P829-binding sites. Because these receptors are ligands for hSSTRS sites was P829 = SST-14 = Tyr'-OCT VIP. expressed at high levels in many different tumors, 99mTc-P829 ap Detection of hSSTR mRNA by Northern Blotting. The presence of hSSTR subtype-specific mRNA in primary tumors (Table 2) and pears as a promising novel tumor-imaging agent. Indeed, our initial imaging studies demonstrate the in vivo binding capacity of ""Tctumor cell lines (Table 3) was analyzed by Northern blotting. As P829 in melanoma and breast cancer patients. can be seen in Fig. 1, C and D, as well as in Tables 2 and 3. primary In the first phase of our study, we analyzed the binding of 99mTctumors and immortalized tumor cell lines all expressed HSSTR3. P829 to a variety of primary human tumors. Similarly to OCT and Also in most primary tumors and tumor cells, expression of Tyr'-OCT (36), this novel SST-analogue bound to virtually all tumor hSSTR2 mRNA as well as hSSTR4 and hSSTRS mRNA was cell membrane fractions, with high affinity and high capacity. The detectable. However, most of the tumors did not express hSSTRl. number of 99mTc-P829 binding sites expressed on these tumors was in Depending on the tumor type, expression of hSSTR subtype mRNA differed with respect to the intensity of Northern blot the range reported earlier for other SSTR ligands (36). Furthermore, the binding of 99mTc-P829 to the membrane fractions could be dis signals (see Tables 2 and 3). placed by unlabeled P829, [ReO]-P829, SST-14, and Tyr'-OCT. This In Vivo Binding of 99mTc-P829 to Primary Tumors or Tumor suggests that 99mTc-P829 acts as a SSTR ligand in many human Mtastases.To test the in vivo applicability of the novel tumor tracer 99mTc-P829, a cohort of patients suffering either from breast tumors tumors. Similar results were obtained with a number of tumor cell (n = 8) or melanomas (n = 6) was subjected to 99mTc-P829 scintig- lines showing high level expression of 99"'Tc-P829 binding sites on intact tumor cells. In contrast, the number of 99mTc-P829 binding sites raphy. In four melanoma patients, we were able to perform compar ative in vitro-in vivo experiments using 99"'Tc-P829. detectable on normal human peripheral blood cells (PMNCs and 99mTc-P829 was well tolerated by all patients. No adverse reactions platelets), as well as normal tissues (see Table 3), was in a lower range. All of these observations suggest that 99mTc-P829 is a prom were observed after peptide injection or during the study period. ising novel imaging agent. Indeed, using 99mTc-P829 as an imaging 99mTc-P829 visualized the primary breast tumor in four of five pa tients (Fig. 4A). 99mTc-P829 also indicated multiple sites of mtasta agent, primary tumors (breast cancers and melanomas) and their ses in three of three patients (Table 4: Fig. 4). urthermore, 99mTc- mtastasescould be detected with high specificity. Whether all pri F mary tumors expressing P829 binding sites can be imaged by 99mTcP829 scintigraphy indicated two primary melanomas (two of two) and multiple sites of melanoma lymph node mtastases,bone, and s.c. P829 remains to be determined in a larger clinical study. A remarkable finding of this study was that almost all tumor cell mtastasesin five of five patients (Table 1). Lymphonodal packages, lines expressed two classes of high-affinity 99n'Tc-P829 binding sites. clinically not evident and unknown prior to scintigraphy were iden This observation confirms our previous data obtained with other tified in three of these patients (Fig. 4B). In four melanoma patients in whom the primary tumor, LNN radiolabeled SST-14 analogues (Tyr'-OCT and SST-14; Ref. 2). With mtastases,or s.c. mtastases were visualized by 99mTc-P829 scintig regard to primary tumor cells, it was not possible to analyze the raphy (Table 1), in vitro receptor binding experiments revealed ex presence of the two classes of binding sites due to the low amount of pression of 99mTc-P829 binding sites. A summary of in vivo receptor available primary tumor tissue. However, in all donors in whom the binding results is provided in Table 1. two binding classes could be analyzed, we were indeed able to detect

Fig. 4. 99mTc-P829 scintigraphy in a breast cancer patient (Table 1. patient I ) and in a melanoma patient (B; Table 1. patient 4). ')9mTc-P829 (500 MBq) significantly concentrated in the 2-cm primary ductal breast cancer (A, right lateral mage prone position; matrix 128 X 128, 1 min postinjection), as well as in multiple sites of melanoma lymph node mtastases in (B. right axilla; C, pelvis; matrix 128 X 128. 2 h postinjection). The tumor sites were still visible at 24 h postinjection in both patients.
the two binding classes. The molecular basis of the heterogeneity of the binding sites remains unknown. One attractive hypothesis is that different types of hSSTR exhibit diverging binding affinity for"mTcP829. In this regard, it is also noteworthy that all of the tumor tissues analyzed expressed multiple types of hSSTR at the mRNA level (see below). Previous data have shown that VIP and SST cross-compete for binding to tumor cell membrane receptors (2). This cross-competition has been observed with primary tumor cells as well as with different tumor cell lines (2). In the present study, we asked whether the hSSTR ligand P829 and VIP cross-compete for binding to primary or immor talized tumor cells. Indeed, cross-competition between these ligands was demonstrable, suggesting that 99nlTc-P829 identifies the common receptor for SST and VIP (20). The next question was whether any of the known hSSTR subytpes or VIPR subtypes mediates this crosscompetition. For this purpose, cDNAs encoding for hSSTR 1 through hSSTRS were transfected into COS7 cells to determine the binding specificity of the radioligand 99mTc-P829. In these experiments, the hSSTR subtypes 2, 3, and 5 were identified as P829 binding sites. The binding of 99mTc-P829 to hSSTR2 was displaced by Tyr3-OCT and SST-14 but not by VIP. In contrast, binding of 99mTc-P829 to hSSTR3 was displaced by Tyr3-OCT and SST-14, as well as by VIP. These data confirm that hSSTR3 is the common receptor for SST-14 and VIP and that 99mTc-P829 is a hSSTR3 ligand. One interesting aspect of our data is that 99mTc-P829 and 123I-VIP bind with higher affinity to hSSTR3 as compared with Tyr'-OCT. The reason for this phenomenon is not known. One possibility could be that the differ ences are due to the different chemical structure of the peptides. In this respect, it is noteworthy that OCT, in contrast to VIP, binds to hSSTR2 and hSSTR5, but not to hSSTRl or hSSTR4. The inability of hSSTRl and hSSTR4 to bind SST analogues such as OCT is consist ent with previous reports (for review, see Ref. 37). The alternative explanation, i.e., an ineffective transfection, seems rather unlikely, because Northern blotting confirmed the expression of these receptors at the RNA level (hSSTRl through hSSTRS). A number of studies have recently been conducted to characterize hSSTR expressed on primary tumors. Using different techniques, hSSTR2 through hSSTRS were found to be expressed in primary human tumors (for a review, see Ref. 24). We have recently shown that among these receptors, hSSTR3 is frequently and strongly ex pressed in a large variety of human tumors, including breast cancers,

intestinal adenocarcinomas, melanomas, and most neuroendocrine tumors using Northern blotting (28). This observation was confirmed in the present study (Fig. 1, C and D). In particular, all investigated tumors and cell lines expressed significant amounts of hSSTR3 mRNA and hSSTR2 mRNA, whereas the other hSSTR subtypes were only weakly expressed or absent. The observation that 99mTc-P829 binds to hSSTRS is of considerable importance both in terms of its potential use as a radioligand and its use as a potential radiopharmaceutical. In conclusion, our data show that 99n'Tc-P829 binds to a number of different human tumors, possibly via hSSTR2, hSSTR5, and the VIP acceptor hSSTR3. Because these receptors are frequently expressed in human tumors and 99mTc labeling is simple and relatively inexpensive and exhibits desirable decay characteristics, 99mTc-P829 may be a most useful tumor-imaging agent in nuclear medicine.

ACKNOWLEDGMENTS

We are grateful to Prof. B. Niederle (Department of Surgery. University of Vienna) for tissue supply, as well as to Dr. W. Acampa for help with the 99mTc-P829 scintigraphy. We are grateful to the technicians Martha Seif, Elisabeth Hangelmann, and Qiong Yang for laboratory assistance.

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