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Towards predictive, preventive, and personalized paediatric surgery
Journal of Pediatric Surgery (2008) 43, 267–273
www.elsevier.com/locate/jpedsurg
Journal of Pediatric Surgery Lecture
Towards predictive, preventive, and personalized paediatric surgery Paul K.H. Tam⁎ Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong Received 28 September 2007; accepted 9 October 2007
Index words: Paediatric surgeons; Genomics; Stem cells; Hirschsprung disease; Personalized medicine
Abstract Future medicine will be revolutionized by genomic and stem cell research, becoming predictive, preventive, and personalized. Despite the smallness of the specialty, paediatric surgery is well placed to play a determining role in this exciting development. First, paediatric surgeons are innovators and leaders. Second, paediatric surgery thrives on the multidisciplinary approach. Third, congenital anomalies provide genetic models for studies of complex diseases. Fourth, morphogenesis underpins basic understanding in development, ageing, cancer, and immunology. The next generation paediatric surgeons must seize the opportunities in large-scale biology research to develop the best future treatment for their patients. © 2008 Published by Elsevier Inc.
1. The genomic and stem cell era The British Association of Paediatric Surgeons was founded in 1953. Its objects are the advancement of study, practice, and research in paediatric surgery. In the same year, Watson and Crick discovered the structure of deoxyribonucleic acid (DNA) and modestly stated that “it has not escaped our notice that the specific paring we have postulated immediately suggests a possible copying mechanism for the genetic material” [1] In fact, the breakthrough has opened up our understanding of molecular biology and genetics in such a way that DNA now underlies every aspect of human health. Understanding gene and protein function will have a profound impact on diagnosis and prediction, prevention, and personalized treatment of diseases. Few will doubt that
Presented at the British Association of Paediatric Surgeons meeting, Edinburgh, Scotland, July 17-20, 2007. ⁎ Tel.: +852 28554850; fax: +852 28173155. E-mail address: [email protected]. 0022-3468/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.jpedsurg.2007.10.012
genomics will bring revolutionary changes in clinical and public health practice. The double breakthroughs of animal cloning by Wilmut and Campbell in 1997 [2] and the first isolation of human embryonic stem cells by Thomson et al in 1998 [3] have generated much excitement both in the scientific community and the general public. Stem cell research offers much hope for cell-replacement therapies for many diseases, and such treatments can be potentially patient specific. The convergence of basic science and clinical medicine has never been closer.
2. Whither paediatric surgery The question is, in this era of genomic and stem cell revolution fuelled by high-cost, fast-moving technologies and competing funding demands, where will paediatric surgeons be? Will we become mere bystanders and fringe players, or can we play a determining role in large-scale biology for the benefit of our patients?
268 The odds are against paediatric surgery. It is a small specialty with small numbers of patients and researchers. To the general public and the policy makers, the diseases in paediatric surgery have low visibility and socioeconomic impact compared with cancer, ageing diseases, infection, etc. As a result, funding opportunities for basic and translational research for our specialty are limited. I will, however, argue that we can overturn the odds. Just as paediatric surgery has now stolen the march in the development of minimal invasive surgery from adult surgery after a slow start, we have several advantages that will allow us to surf ahead on the crest of the waves of biomedical research. My arguments are as follows: 1. Surgeons in general and paediatric surgeons in particular are innovators and leaders. 2. Paediatric surgery thrives on multidisciplinary approach. 3. Congenital anomalies provide genetic models for studies of complex diseases. 4. Morphogenesis underpins basic understanding not only in development, but also in ageing, cancer, and immunology. I contend that there are many examples to support the arguments; but without attempting to be exhaustive and comprehensive, I will illustrate some arguments with personal experiences.
3. Innovation and leadership Surgeons are innovators and problem solvers. The history of surgery is annotated with breakthrough discoveries. The only medical advance to make it into Life Magazine's top 10 most important advances of the previous millennium was the germ theory, founded on the works of a surgeon, Joseph Lister, on antisepsis. No less than 9 surgeons were awarded the Nobel Prize in Medicine and Physiology from 1901 to 2005 for works ranging from understanding the thyroid, to discoveries of cancer treatment, and to transplantation [4]. In the recent decades, one of the most important biomedical discoveries, angiogenesis, was made by a paediatric surgeon, Dr Judah Folkman [5]. Modern biological research is increasingly team based. Paediatric surgeons are good team players and effective team leaders. There are many examples of paediatric surgeons establishing themselves as leaders beyond their specialty. Dr CE Koop was Surgeon General of the United States, the nation's top administrative health post, in the 1980s. Surgical Associations and Colleges around the world have elected paediatric surgeons as their presidents, for example, J Grosfeld (American Surgical Association), K Anderson (American College of Surgeons), J Orr (Royal College of Surgeons, Edinburgh), A Kolbe (Royal Australasian College of Surgeons), etc. Universities and hospitals have appointed paediatric surgeons as heads, for example, F Cigarroa (Chancellor, University of
P.K.H. Tam Texas), WJ Chen (President, National Taiwan University), P Tam (Vice President, the University of Hong Kong). Leadership outside the specialty can bring benefit to paediatric surgery because policy makers will understand better children's needs and afford paediatric surgeons a more level playing field in the competition for research funding.
4. Multidisciplinary research The “big science” nowadays is dominated by the multidisciplinary approach, an approach that many conventional scientists are still unaccustomed to. Paediatric surgeons have always cherished the multidisciplinary approach in clinical care and therefore are well positioned to extend this attitude to research. In the past decade, the most exciting research projects I have participated in have been multidisciplinary and have involved the establishment of expensive, cuttingedge technologies that single laboratories cannot afford.
5. Centre of human development and birth defect I returned to the University of Hong Kong in 1996 and encountered all the difficulties that a researcher in a small clinical discipline would face, especially the lack of a critical mass of researchers to make an impact. However, as a paediatric surgeon, I was keen to reach out; and it soon became clear to me that developmental biologists similarly felt isolated. The clinicians have the problems, whereas the scientists have the tools. We founded the Centre of Human Development and Birth Defects in 1999 with the aim of linking clinicians and scientists to explore gene function in development and diseases. Model systems including mouse, rat, chick worm, fish, and frog were established. Transgenic and knockout mice were created. A congenital malformation registry with protocol-based sample collection was set up. Through regular meetings, research collaboration increased substantially. With increasing research productivity, the Centre expanded in size and funding, acquiring recognition as one of the University's strategic research themes and an Area of Excellence in Hong Kong. The Centre is now well placed to play a leading role in stem cell biology and regenerative medicine research.
6. Genome research centre In 2001, the University of Hong Kong identified genomics as a major strategic research direction of the University and allocated a sum of HK$120 million (≈US$15 million) to establish the Genome Research Centre. The Centre provides the expertise and infrastructure in genomics, proteomics, and bioinformatics for scientists and clinicians to apply them for studies to understand and treat diseases. This signals the move
Towards predictive, preventive, and personalized paediatric surgery from individual investigator–based, low-budget, low-throughput studies to multidisciplinary team–based, high-technology, large-scale biology research. As Deputy Chairman of the Steering Committee, I oversaw the establishment of core capabilities of microarray, genotyping, genetic sequencing, proteomics, and bioinformatics, and became familiar with those powerful tools (Fig. 1).
7. HapMap: predictive, preventive, and personalized medicine? The establishment of the Genome Research Centre enabled the University of Hong Kong to participate in the
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International HapMap Project. The completion of the Human Genome Project tin 2001 was lauded as the biomedical equivalent of “putting men on the moon.” Humans are genetically 99.9% identical. The genome project gave us that common DNA code. However, it is the remaining 0.1% that makes us different, for example, in our predisposition to diseases and response to treatments. The International HapMap Project aims to provide a detailed map of common genetic variation of the human genome in diverse populations to facilitate systematic genetic association studies on complex disorders [6]. The project focuses on DNA markers called single nucleotide polymorphisms (SNPs), which are variations at the single base level. Long segments of DNA are inherited together as unbroken blocks (haplotypes) and
Fig. 1 Genomic technologies at the Genome Research Centre. (A) High-throughput liquid handling system. (B) Detailed view of a 96-well– plate format liquid handling system. (C) Mass spectrometer used in the SEQUENOM MassARRAY genotyping technology. (D) ABI 3730xl Genetic Analyzer used for high-throughput direct sequencing and gene scan. (E) GeneChip fluidics station used when processing Affymetrix chips containing probes for 1,800,000 genetic markers (chip on the top right). (F) API 2000 triple quadruple mass spectrometer directly coupled to a Perkin Elmer series 200 high-performance liquid chromatography system used in the separation, characterization, and quantification of biological samples.
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can be identified by a handful of SNPs. A catalog of haplotypes offers a shortcut to find variant genes because instead of sequencing the entire genome, investigators could just ask which haplotypes are more common in patients with a particular disease, greatly narrowing the search for causative mutations. Phase I of the project aimed to achieve density of less than 1 SNP per 5000 base pairs in 269 individuals from populations of European (90), Asian (89), and African (90) origins. The US$138 million project was undertaken by an International Consortium involving some 200 investigators. Genotyping work was allocated to centres in the United States (30%), United Kingdom (25%), Japan (25%), Canada (10%), and China (10%); our group was responsible for the short arm of chromosome 3, a region equivalent to 2.5% of the genome. The Genome Research Centre at the University of Hong Kong had to develop highthroughput, large-scale genotyping capacities and meet quality controls and assurance on tight deadlines in synchrony with other advanced centres. On 27 October 2005, 3 years after the launch of the project, the Consortium announced the completion of the haplotype map to much public acclaim. There were more than 450 news reports worldwide. According to Newsweek, the reason for all the excitement is simple: “the HapMap may finally usher in the era of preventive medicine.” For BBC News, the “Gene Map points to personal drugs.” The database, published in Nature [7] (Fig. 2), revealed 3 major findings: the generality of hotspots of recombination, long segments of strong linkage disequilibrium, and limited haplotype diversity. Using HapMap, it is thus possible to
Fig. 2
extract extensive information about genomic variation without complete resequencing and enable genome-wide association studies to be carried out efficiently through selection of appropriate tag SNPs. A higher-density map with 3.4 million SNPs has now been completed. This improves genome-wide association studies, reveals novel aspects of structure of linkage disequilibrium, and provides new information about influence of natural selection on protein-changing variants [8]. Participation in the HapMap project has been a valuable experience for me. Apart from publications and service to humankind, new insights were gained: (1) Technology development drives cost reduction—within 2 years, the cost of high-throughput automated genotyping has fallen from 50¢ to 10¢ per SNP. (2) To handle the massive data generated, a new breed of specialists in bioinformatics has to be developed. (3) The collaborative experience of 200 investigators from diverse backgrounds enriches me both at personal and scientific levels. (4) Genome-wide association studies of complex diseases are now feasible, bringing hopes for prevention and new treatment; for example, 4 novel risk loci have been identified for type 2 diabetes mellitus using this approach [9].
8. Congenital anomaly: a genetic model Congenital anomalies such as Hirschsprung disease (HSCR) serve as valuable models for the genetic dissection of complex diseases. The observation of a deletion in
Completion of the HapMap project.
Towards predictive, preventive, and personalized paediatric surgery 10q11.21 in 2 HSCR patients [10,11] allowed the search for an HSCR gene to be focused in that chromosomal region. Linkage studies, positional cloning, and mutation analysis led to the identification of RET as the first HSCR gene (see review, Tam and Garcia-Barcelo [12]). Biological studies including knockout mouse models led to better understanding of the RET signaling pathway and identification of GDNF and NRTN as additional HSCR genes. Using similar strategies, further gene discoveries have been made. To date, at least 9 genes have been identified in HSCR (Table 1, reproduced from Tam and Garcia-Barcelo review [12]). Despite these discoveries, the enigma of HSCR as an oligogenic disorder has not been totally unravelled. Coding sequence mutations of known HSCR genes still account for less than 30% of HSCR patients. Therefore, it is likely that (a) noncoding sequence mutations of HSCR genes are important, (b) other HSCR genes are to be found, and (c) genetic risks may be synergistic. For example, we recently found that whereas the RET AA genotype increases HSCR risk by an odds ratio of 7.98 (95% confidence interval [CI], 3.64-17.60) and PHOX2B AA genotype increases HSCR risk by an odds ratio of 1.56 (95% CI, 0.82-2.75), the combined genotypes increase HSCR risk by an odds ratio of 11.72 (95% CI, 6.35-21.55), suggesting a synergistic effect over and above the additive affect [13]. Because of the heterogeneity of HSCR and the relative low incidence of the condition, a larger sample size is required to advance HSCR studies. An International HSCR Consortium comprising 6 centres in 3 continents was formed in 2004, and 864 HSCR families were studied [14] (Fig. 3). Table 1
Gene involved in Hirschsprung's disease (HSCR)
Gene
% of patients Phenotype in mutants with mutations
RET
≈50% familial cases; 7%-35% sporadic cases 1 case 1 case ≈7% b5% 1 case
GDNF* NRTN* EDNRB EDN3 ECE-1
SOX10 17 cases ZFHX1B b1%
PHOX2B 12 cases 3 cases
HSCR either isolated or syndromic HSCR HSCR HSCR or WS4 HSCR or WS4 Syndromic HSCR (craniofacial and cardiac defects) WS4 Syndromic HSCR (mental retardation and facial dysmorphism) Haddad syndrome (CCHS with aganglionosis) Neuroblastoma associated with HSCR
WS4, Waardenburg-shah syndrome; CCHS, congenital central hypoventilation syndrome. * Mutations found in individuals who also had mutations in the RET gene.
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The initial findings (a) showed a higher prevalence of a noncoding sequence “T” RET mutation in the male population, short-segment HSCR patients, and sporadic HSCR patients (unpublished data) and (b) confirmed our previous results reporting that the higher frequency of RET noncoding region mutations in the Chinese population could explain the higher incidence of HSCR in this population [15]. In searching for new HSCR genes, there is little doubt that the HapMap data and the new gene chip technology will be most helpful. Our group has started a genome-wide SNP association study comparing 500,000 SNP loci in HSCR patients and healthy individuals. Initial findings have confirmed that RET remains the most important locus for HSCR, and several novel susceptibility loci have been identified (unpublished data). Experience obtained in analyzing the massive data from an oligogenic disorder such as HSCR is likely to be of relevance in studies of more complex diseases, such as adult hypertension, cancers, diabetes, etc.
9. Morphogenesis: a building block for disease treatment Morphogenesis has been the cornerstone in our understanding of the pathogenesis of many paediatric surgical diseases; but in recent years, this understanding has found applications in the rapidly growing field of stem cell biology and regenerative medicine. Funding opportunities for stem cell research have escalated exponentially in recent years; for example, in 2005, federal funding in the United States amounted to $550 million (the State of California additionally earmarked a $3 billion budget over 10 years,), and European Union funding totaled $170 million in 3 years [16]. Although the potentials of procuring cells that are pluripotent but can be directed to differentiate and proliferate to replace lost, damaged, or diseased tissues/organs are immediately apparent, understanding the biological processes of maintaining “stemness,” proliferation, and differentiation is equally important and can underpin new knowledge of mechanisms of a wide variety of diseases beyond childhood. Morphogenesis of neural crest has been the subject of interest of paediatric surgeons for many decades. Neural crest cells (NCCs) migrate from the neural tube to their target sites in embryonic development and differentiate into a wide variety of cell types; that is, they possess stem cell properties such as self renewal and differentiation into progenitors of multiple lineages. Hirschsprung disease is characterized by an absence of ganglion in the distal gut. Although the pathology essentially involves only one component of the gut, that is, the enteric nervous system, current treatment requires removal of the entire abnormal enteric nervous system–bearing gut and a pull through of proximal ganglionic gut.
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Fig. 3
International HSCR Consortium study.
Cell replacement therapy could restore the deficient neural function and gut motility while preserving the integrity of the patient's gut. As an indication of the feasibility in this approach, our laboratory has recently successfully isolated neural crest stem cells from the “normal” proximal gut of a 4-month-old HSCR patient and maintained these in a culture system (Fig. 4). Many fundamental biological issues, however, remain to be solved for stem cell therapy to become practical in clinic. To better understand the trophic factors regulating NCC development, our group cultured dissociated cells of E11.5 mouse embryonic guts in NCC medium. The NCCs develop into neurospheres that can self renew. The morphogen sonic hedgehog (Shh) was found to promote NCC proliferation and restrict the GDNF-induced NCC migration [17]. However, the effects of morphogens are not restricted to the embryonic period. Signaling pathways associated with
embryogenesis such as Shh are also involved in peripheral immunity and tissue remodeling. In a rat orthotopic small bowel transplantation model, we showed that anti-shh treatment significantly prolonged graft survival (treated vs controls: 116 vs 77.5 days) and reduced chronic rejection [18]. Collagen deposition, mesenteric vascular occlusion, and vascular endothelial growth factor expression were significantly reduced in recipients of the anti-shh. The association of morphogenesis with oncogenesis is even more apparent. Loss of function mutations of the RET gene results in HSCR, whereas gain of function mutations of RET is associated with neuroendocrine tumours including multiple endocrine neoplasias and medullary thyroid cancer. Inappropriate activation of the Shh pathway is associated with a variety of childhood and adult cancers of the brain and skin, and possibly the gastrointestinal tract. We have shown that prokineticin-1 maintains proliferation
Fig. 4 Cell replacement as a possible treatment of HSCR. Neural crest stem cells isolated from the normal proximal gut of a 4-month-old HSCR female patient were isolated and cultured for several days.
Towards predictive, preventive, and personalized paediatric surgery and differentiation of enteric NCC during embryogenesis [19], whereas aberrant endocrine gland–derived vascular endothelial growth factor/prokineticin-1 signaling favors postnatal neuroblastoma progression [20]. There is growing evidence for the hierarchical model of carcinogenesis that suggests that only a small group of cells, known as cancer stem cells, is able to initiate and maintain a cancerous growth [21]. Identifying stem cell markers could provide potential new targets for chemotherapy and revolutionize cancer treatment. There are many hurdles in the application of stem cells in therapy and tissue regeneration, including the choice of stem cells (embryonic stem cells, fetal stem cells, cord blood or somatic stem cells), immune rejection, long-term survival and functions, teratoma formation, controlled differentiation, routes of administration, and ethical issues. The obstacles, however, are not insurmountable. For example, the immune rejection of nonself stem cells may be overcome by modulating/deleting the patient's immune system during therapy, providing genetically modified universal donor cells, or establishing a stem cell bank for good genetic match of the most common human leukocyte antigen types. Lastly, somatic cell nuclear transfer of a patient's DNA into a donated egg epitomizes personalized medicine.
10. The future “Change is inevitable. Change is constant.” Benjamin Disraeli “It is time for a new generation of leadership, to cope with a new problem and new opportunities. For there is a new world to be won.” JF Kennedy. In June 2007, the full genome of James Watson, codiscoverer of the structure of DNA in 1953, was resequenced, marking the gateway to an impending era of personalized genomic medicine. The importance of genomics to future medicine cannot be overemphasized, and so too are stem cells. As advocates for sick children, we must strive to provide our patients with the best possible treatment. Paediatric surgeons should therefore actively participate in the genomic and stem cell revolution to ensure that future paediatric surgery will be predictive, preventive, and personalized.
Acknowledgment I thank all my colleagues who have generously contributed to the work described, especially Drs VCH Lui, MM Garcia-Barcelo, E Ngan, Y Chen, KKY Wong, X Miao, and Ms MT So, and colleagues at the Genome Research Centre, the University of Hong Kong. The studies were supported by grants from the Research Grants Council
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Hong Kong and the University of Hong Kong. Many scientists and pediatric surgeons around the world have published relevant important papers that are not cited in this article because of space limitations.
References [1] Watson JD, Crick FHC. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature 1953;171:737. [2] Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810-3. [3] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-7. [4] Cosimi AB. Surgeons and the Nobel Prize. Arch Surg 2006;141:340-8. [5] Folkman J. Is angiogenesis an organizing principle in biology and medicine? J Pediatr Surg 2007;42:1-11. [6] The International HapMap Consortium. The International HapMap Project. Nature 2003;426:789-96. [7] The International HapMap Consortium. A haplotype map of the human genome. Nature 2005;437:1299-320. [8] The International HapMap Consortium. The phase II HapMap. Nature (submitted). [9] Sladek R, Rocheleau G, Rung J, et al. A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature 2007;445:881-5. [10] Martucciello G, Biocchini M, Dodero P, et al. Total colonic aganglionosis associated with interstitial deletion of the long arm of chromosome 10. Pediatr Surg Int 1992;7:308. [11] Fewtrell MS, Tam PK, Thomson AH, et al. Hirschsprung's disease associated with a deletion of chromosome 10 (q11.2q21.2): a further link with the neurocristopathies? J Med Genet 1994;31:325-7. [12] Tam PKH, Garcia-Barcelo M. Molecular genetics of Hirschsprung's disease. Seminars in Pediatr Surg 2004;13:236-48. [13] Miao XP, Garcio-Barcelo MM, So MT, et al. Role of RET and PHOX2B gene polymorphisms in risk of Hirschsprung's disease in Chinese population. Gut 2007;56:736. [14] Tam PKH, Garcia-Barcelo MM, Tullio-Pelet A, et al. The International Hirschsprung's disease Consortium study: differential liabilities of coding and non-coding mutations in complex disease. Proceedings of the 53rd Annual International Congress of the British Association of Paediatric Surgeons, Stockholm, Sweden, 18-21 July; 2006. p. 49. [15] Garcia-Barcelo M, Sham MH, Lui VC, et al. Chinese patients with sporadic Hirschsprung's disease are predominantly represented by a single RET haplotype. J Med Genet 2003;40:e122. [16] Beardsley S. A world of approaches to stem cells. Sci Am 2005:A20. [17] Tian L, Lu LW, Yuan ZW, et al. Acceleration of apoptosis in CD4+CD8+ thymocytes by rapamycin accompanied with increased CD4+CD25+ T cells in the periphery. Transplantation 2004;77: 183-9. [18] Chen Y, Li XS, Tina T, et al. Inhibition of sonic hedgehog signalling reduces chronic rejection and prolongs allograft survival in a rat orthotopic small bowel transplantation model. Transplantation 2007; 83:1351-7. [19] Ngan ESW, Lee KY, Sit FYL, et al. Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells. Biochim Biophys Acta 2007;1773:536-45. [20] Ngan ESW, Sit FYL, Lee KL, et al. Implications of endocrine gland–derived vascular endothelial growth factor/prokineticin-1 signaling in human neuroblastoma progression. Clin Cancer Res 2007;13:868-75. [21] Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell 2006;124:1111-5.
www.elsevier.com/locate/jpedsurg
Journal of Pediatric Surgery Lecture
Towards predictive, preventive, and personalized paediatric surgery Paul K.H. Tam⁎ Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong Received 28 September 2007; accepted 9 October 2007
Index words: Paediatric surgeons; Genomics; Stem cells; Hirschsprung disease; Personalized medicine
Abstract Future medicine will be revolutionized by genomic and stem cell research, becoming predictive, preventive, and personalized. Despite the smallness of the specialty, paediatric surgery is well placed to play a determining role in this exciting development. First, paediatric surgeons are innovators and leaders. Second, paediatric surgery thrives on the multidisciplinary approach. Third, congenital anomalies provide genetic models for studies of complex diseases. Fourth, morphogenesis underpins basic understanding in development, ageing, cancer, and immunology. The next generation paediatric surgeons must seize the opportunities in large-scale biology research to develop the best future treatment for their patients. © 2008 Published by Elsevier Inc.
1. The genomic and stem cell era The British Association of Paediatric Surgeons was founded in 1953. Its objects are the advancement of study, practice, and research in paediatric surgery. In the same year, Watson and Crick discovered the structure of deoxyribonucleic acid (DNA) and modestly stated that “it has not escaped our notice that the specific paring we have postulated immediately suggests a possible copying mechanism for the genetic material” [1] In fact, the breakthrough has opened up our understanding of molecular biology and genetics in such a way that DNA now underlies every aspect of human health. Understanding gene and protein function will have a profound impact on diagnosis and prediction, prevention, and personalized treatment of diseases. Few will doubt that
Presented at the British Association of Paediatric Surgeons meeting, Edinburgh, Scotland, July 17-20, 2007. ⁎ Tel.: +852 28554850; fax: +852 28173155. E-mail address: [email protected]. 0022-3468/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.jpedsurg.2007.10.012
genomics will bring revolutionary changes in clinical and public health practice. The double breakthroughs of animal cloning by Wilmut and Campbell in 1997 [2] and the first isolation of human embryonic stem cells by Thomson et al in 1998 [3] have generated much excitement both in the scientific community and the general public. Stem cell research offers much hope for cell-replacement therapies for many diseases, and such treatments can be potentially patient specific. The convergence of basic science and clinical medicine has never been closer.
2. Whither paediatric surgery The question is, in this era of genomic and stem cell revolution fuelled by high-cost, fast-moving technologies and competing funding demands, where will paediatric surgeons be? Will we become mere bystanders and fringe players, or can we play a determining role in large-scale biology for the benefit of our patients?
268 The odds are against paediatric surgery. It is a small specialty with small numbers of patients and researchers. To the general public and the policy makers, the diseases in paediatric surgery have low visibility and socioeconomic impact compared with cancer, ageing diseases, infection, etc. As a result, funding opportunities for basic and translational research for our specialty are limited. I will, however, argue that we can overturn the odds. Just as paediatric surgery has now stolen the march in the development of minimal invasive surgery from adult surgery after a slow start, we have several advantages that will allow us to surf ahead on the crest of the waves of biomedical research. My arguments are as follows: 1. Surgeons in general and paediatric surgeons in particular are innovators and leaders. 2. Paediatric surgery thrives on multidisciplinary approach. 3. Congenital anomalies provide genetic models for studies of complex diseases. 4. Morphogenesis underpins basic understanding not only in development, but also in ageing, cancer, and immunology. I contend that there are many examples to support the arguments; but without attempting to be exhaustive and comprehensive, I will illustrate some arguments with personal experiences.
3. Innovation and leadership Surgeons are innovators and problem solvers. The history of surgery is annotated with breakthrough discoveries. The only medical advance to make it into Life Magazine's top 10 most important advances of the previous millennium was the germ theory, founded on the works of a surgeon, Joseph Lister, on antisepsis. No less than 9 surgeons were awarded the Nobel Prize in Medicine and Physiology from 1901 to 2005 for works ranging from understanding the thyroid, to discoveries of cancer treatment, and to transplantation [4]. In the recent decades, one of the most important biomedical discoveries, angiogenesis, was made by a paediatric surgeon, Dr Judah Folkman [5]. Modern biological research is increasingly team based. Paediatric surgeons are good team players and effective team leaders. There are many examples of paediatric surgeons establishing themselves as leaders beyond their specialty. Dr CE Koop was Surgeon General of the United States, the nation's top administrative health post, in the 1980s. Surgical Associations and Colleges around the world have elected paediatric surgeons as their presidents, for example, J Grosfeld (American Surgical Association), K Anderson (American College of Surgeons), J Orr (Royal College of Surgeons, Edinburgh), A Kolbe (Royal Australasian College of Surgeons), etc. Universities and hospitals have appointed paediatric surgeons as heads, for example, F Cigarroa (Chancellor, University of
P.K.H. Tam Texas), WJ Chen (President, National Taiwan University), P Tam (Vice President, the University of Hong Kong). Leadership outside the specialty can bring benefit to paediatric surgery because policy makers will understand better children's needs and afford paediatric surgeons a more level playing field in the competition for research funding.
4. Multidisciplinary research The “big science” nowadays is dominated by the multidisciplinary approach, an approach that many conventional scientists are still unaccustomed to. Paediatric surgeons have always cherished the multidisciplinary approach in clinical care and therefore are well positioned to extend this attitude to research. In the past decade, the most exciting research projects I have participated in have been multidisciplinary and have involved the establishment of expensive, cuttingedge technologies that single laboratories cannot afford.
5. Centre of human development and birth defect I returned to the University of Hong Kong in 1996 and encountered all the difficulties that a researcher in a small clinical discipline would face, especially the lack of a critical mass of researchers to make an impact. However, as a paediatric surgeon, I was keen to reach out; and it soon became clear to me that developmental biologists similarly felt isolated. The clinicians have the problems, whereas the scientists have the tools. We founded the Centre of Human Development and Birth Defects in 1999 with the aim of linking clinicians and scientists to explore gene function in development and diseases. Model systems including mouse, rat, chick worm, fish, and frog were established. Transgenic and knockout mice were created. A congenital malformation registry with protocol-based sample collection was set up. Through regular meetings, research collaboration increased substantially. With increasing research productivity, the Centre expanded in size and funding, acquiring recognition as one of the University's strategic research themes and an Area of Excellence in Hong Kong. The Centre is now well placed to play a leading role in stem cell biology and regenerative medicine research.
6. Genome research centre In 2001, the University of Hong Kong identified genomics as a major strategic research direction of the University and allocated a sum of HK$120 million (≈US$15 million) to establish the Genome Research Centre. The Centre provides the expertise and infrastructure in genomics, proteomics, and bioinformatics for scientists and clinicians to apply them for studies to understand and treat diseases. This signals the move
Towards predictive, preventive, and personalized paediatric surgery from individual investigator–based, low-budget, low-throughput studies to multidisciplinary team–based, high-technology, large-scale biology research. As Deputy Chairman of the Steering Committee, I oversaw the establishment of core capabilities of microarray, genotyping, genetic sequencing, proteomics, and bioinformatics, and became familiar with those powerful tools (Fig. 1).
7. HapMap: predictive, preventive, and personalized medicine? The establishment of the Genome Research Centre enabled the University of Hong Kong to participate in the
269
International HapMap Project. The completion of the Human Genome Project tin 2001 was lauded as the biomedical equivalent of “putting men on the moon.” Humans are genetically 99.9% identical. The genome project gave us that common DNA code. However, it is the remaining 0.1% that makes us different, for example, in our predisposition to diseases and response to treatments. The International HapMap Project aims to provide a detailed map of common genetic variation of the human genome in diverse populations to facilitate systematic genetic association studies on complex disorders [6]. The project focuses on DNA markers called single nucleotide polymorphisms (SNPs), which are variations at the single base level. Long segments of DNA are inherited together as unbroken blocks (haplotypes) and
Fig. 1 Genomic technologies at the Genome Research Centre. (A) High-throughput liquid handling system. (B) Detailed view of a 96-well– plate format liquid handling system. (C) Mass spectrometer used in the SEQUENOM MassARRAY genotyping technology. (D) ABI 3730xl Genetic Analyzer used for high-throughput direct sequencing and gene scan. (E) GeneChip fluidics station used when processing Affymetrix chips containing probes for 1,800,000 genetic markers (chip on the top right). (F) API 2000 triple quadruple mass spectrometer directly coupled to a Perkin Elmer series 200 high-performance liquid chromatography system used in the separation, characterization, and quantification of biological samples.
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can be identified by a handful of SNPs. A catalog of haplotypes offers a shortcut to find variant genes because instead of sequencing the entire genome, investigators could just ask which haplotypes are more common in patients with a particular disease, greatly narrowing the search for causative mutations. Phase I of the project aimed to achieve density of less than 1 SNP per 5000 base pairs in 269 individuals from populations of European (90), Asian (89), and African (90) origins. The US$138 million project was undertaken by an International Consortium involving some 200 investigators. Genotyping work was allocated to centres in the United States (30%), United Kingdom (25%), Japan (25%), Canada (10%), and China (10%); our group was responsible for the short arm of chromosome 3, a region equivalent to 2.5% of the genome. The Genome Research Centre at the University of Hong Kong had to develop highthroughput, large-scale genotyping capacities and meet quality controls and assurance on tight deadlines in synchrony with other advanced centres. On 27 October 2005, 3 years after the launch of the project, the Consortium announced the completion of the haplotype map to much public acclaim. There were more than 450 news reports worldwide. According to Newsweek, the reason for all the excitement is simple: “the HapMap may finally usher in the era of preventive medicine.” For BBC News, the “Gene Map points to personal drugs.” The database, published in Nature [7] (Fig. 2), revealed 3 major findings: the generality of hotspots of recombination, long segments of strong linkage disequilibrium, and limited haplotype diversity. Using HapMap, it is thus possible to
Fig. 2
extract extensive information about genomic variation without complete resequencing and enable genome-wide association studies to be carried out efficiently through selection of appropriate tag SNPs. A higher-density map with 3.4 million SNPs has now been completed. This improves genome-wide association studies, reveals novel aspects of structure of linkage disequilibrium, and provides new information about influence of natural selection on protein-changing variants [8]. Participation in the HapMap project has been a valuable experience for me. Apart from publications and service to humankind, new insights were gained: (1) Technology development drives cost reduction—within 2 years, the cost of high-throughput automated genotyping has fallen from 50¢ to 10¢ per SNP. (2) To handle the massive data generated, a new breed of specialists in bioinformatics has to be developed. (3) The collaborative experience of 200 investigators from diverse backgrounds enriches me both at personal and scientific levels. (4) Genome-wide association studies of complex diseases are now feasible, bringing hopes for prevention and new treatment; for example, 4 novel risk loci have been identified for type 2 diabetes mellitus using this approach [9].
8. Congenital anomaly: a genetic model Congenital anomalies such as Hirschsprung disease (HSCR) serve as valuable models for the genetic dissection of complex diseases. The observation of a deletion in
Completion of the HapMap project.
Towards predictive, preventive, and personalized paediatric surgery 10q11.21 in 2 HSCR patients [10,11] allowed the search for an HSCR gene to be focused in that chromosomal region. Linkage studies, positional cloning, and mutation analysis led to the identification of RET as the first HSCR gene (see review, Tam and Garcia-Barcelo [12]). Biological studies including knockout mouse models led to better understanding of the RET signaling pathway and identification of GDNF and NRTN as additional HSCR genes. Using similar strategies, further gene discoveries have been made. To date, at least 9 genes have been identified in HSCR (Table 1, reproduced from Tam and Garcia-Barcelo review [12]). Despite these discoveries, the enigma of HSCR as an oligogenic disorder has not been totally unravelled. Coding sequence mutations of known HSCR genes still account for less than 30% of HSCR patients. Therefore, it is likely that (a) noncoding sequence mutations of HSCR genes are important, (b) other HSCR genes are to be found, and (c) genetic risks may be synergistic. For example, we recently found that whereas the RET AA genotype increases HSCR risk by an odds ratio of 7.98 (95% confidence interval [CI], 3.64-17.60) and PHOX2B AA genotype increases HSCR risk by an odds ratio of 1.56 (95% CI, 0.82-2.75), the combined genotypes increase HSCR risk by an odds ratio of 11.72 (95% CI, 6.35-21.55), suggesting a synergistic effect over and above the additive affect [13]. Because of the heterogeneity of HSCR and the relative low incidence of the condition, a larger sample size is required to advance HSCR studies. An International HSCR Consortium comprising 6 centres in 3 continents was formed in 2004, and 864 HSCR families were studied [14] (Fig. 3). Table 1
Gene involved in Hirschsprung's disease (HSCR)
Gene
% of patients Phenotype in mutants with mutations
RET
≈50% familial cases; 7%-35% sporadic cases 1 case 1 case ≈7% b5% 1 case
GDNF* NRTN* EDNRB EDN3 ECE-1
SOX10 17 cases ZFHX1B b1%
PHOX2B 12 cases 3 cases
HSCR either isolated or syndromic HSCR HSCR HSCR or WS4 HSCR or WS4 Syndromic HSCR (craniofacial and cardiac defects) WS4 Syndromic HSCR (mental retardation and facial dysmorphism) Haddad syndrome (CCHS with aganglionosis) Neuroblastoma associated with HSCR
WS4, Waardenburg-shah syndrome; CCHS, congenital central hypoventilation syndrome. * Mutations found in individuals who also had mutations in the RET gene.
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The initial findings (a) showed a higher prevalence of a noncoding sequence “T” RET mutation in the male population, short-segment HSCR patients, and sporadic HSCR patients (unpublished data) and (b) confirmed our previous results reporting that the higher frequency of RET noncoding region mutations in the Chinese population could explain the higher incidence of HSCR in this population [15]. In searching for new HSCR genes, there is little doubt that the HapMap data and the new gene chip technology will be most helpful. Our group has started a genome-wide SNP association study comparing 500,000 SNP loci in HSCR patients and healthy individuals. Initial findings have confirmed that RET remains the most important locus for HSCR, and several novel susceptibility loci have been identified (unpublished data). Experience obtained in analyzing the massive data from an oligogenic disorder such as HSCR is likely to be of relevance in studies of more complex diseases, such as adult hypertension, cancers, diabetes, etc.
9. Morphogenesis: a building block for disease treatment Morphogenesis has been the cornerstone in our understanding of the pathogenesis of many paediatric surgical diseases; but in recent years, this understanding has found applications in the rapidly growing field of stem cell biology and regenerative medicine. Funding opportunities for stem cell research have escalated exponentially in recent years; for example, in 2005, federal funding in the United States amounted to $550 million (the State of California additionally earmarked a $3 billion budget over 10 years,), and European Union funding totaled $170 million in 3 years [16]. Although the potentials of procuring cells that are pluripotent but can be directed to differentiate and proliferate to replace lost, damaged, or diseased tissues/organs are immediately apparent, understanding the biological processes of maintaining “stemness,” proliferation, and differentiation is equally important and can underpin new knowledge of mechanisms of a wide variety of diseases beyond childhood. Morphogenesis of neural crest has been the subject of interest of paediatric surgeons for many decades. Neural crest cells (NCCs) migrate from the neural tube to their target sites in embryonic development and differentiate into a wide variety of cell types; that is, they possess stem cell properties such as self renewal and differentiation into progenitors of multiple lineages. Hirschsprung disease is characterized by an absence of ganglion in the distal gut. Although the pathology essentially involves only one component of the gut, that is, the enteric nervous system, current treatment requires removal of the entire abnormal enteric nervous system–bearing gut and a pull through of proximal ganglionic gut.
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Fig. 3
International HSCR Consortium study.
Cell replacement therapy could restore the deficient neural function and gut motility while preserving the integrity of the patient's gut. As an indication of the feasibility in this approach, our laboratory has recently successfully isolated neural crest stem cells from the “normal” proximal gut of a 4-month-old HSCR patient and maintained these in a culture system (Fig. 4). Many fundamental biological issues, however, remain to be solved for stem cell therapy to become practical in clinic. To better understand the trophic factors regulating NCC development, our group cultured dissociated cells of E11.5 mouse embryonic guts in NCC medium. The NCCs develop into neurospheres that can self renew. The morphogen sonic hedgehog (Shh) was found to promote NCC proliferation and restrict the GDNF-induced NCC migration [17]. However, the effects of morphogens are not restricted to the embryonic period. Signaling pathways associated with
embryogenesis such as Shh are also involved in peripheral immunity and tissue remodeling. In a rat orthotopic small bowel transplantation model, we showed that anti-shh treatment significantly prolonged graft survival (treated vs controls: 116 vs 77.5 days) and reduced chronic rejection [18]. Collagen deposition, mesenteric vascular occlusion, and vascular endothelial growth factor expression were significantly reduced in recipients of the anti-shh. The association of morphogenesis with oncogenesis is even more apparent. Loss of function mutations of the RET gene results in HSCR, whereas gain of function mutations of RET is associated with neuroendocrine tumours including multiple endocrine neoplasias and medullary thyroid cancer. Inappropriate activation of the Shh pathway is associated with a variety of childhood and adult cancers of the brain and skin, and possibly the gastrointestinal tract. We have shown that prokineticin-1 maintains proliferation
Fig. 4 Cell replacement as a possible treatment of HSCR. Neural crest stem cells isolated from the normal proximal gut of a 4-month-old HSCR female patient were isolated and cultured for several days.
Towards predictive, preventive, and personalized paediatric surgery and differentiation of enteric NCC during embryogenesis [19], whereas aberrant endocrine gland–derived vascular endothelial growth factor/prokineticin-1 signaling favors postnatal neuroblastoma progression [20]. There is growing evidence for the hierarchical model of carcinogenesis that suggests that only a small group of cells, known as cancer stem cells, is able to initiate and maintain a cancerous growth [21]. Identifying stem cell markers could provide potential new targets for chemotherapy and revolutionize cancer treatment. There are many hurdles in the application of stem cells in therapy and tissue regeneration, including the choice of stem cells (embryonic stem cells, fetal stem cells, cord blood or somatic stem cells), immune rejection, long-term survival and functions, teratoma formation, controlled differentiation, routes of administration, and ethical issues. The obstacles, however, are not insurmountable. For example, the immune rejection of nonself stem cells may be overcome by modulating/deleting the patient's immune system during therapy, providing genetically modified universal donor cells, or establishing a stem cell bank for good genetic match of the most common human leukocyte antigen types. Lastly, somatic cell nuclear transfer of a patient's DNA into a donated egg epitomizes personalized medicine.
10. The future “Change is inevitable. Change is constant.” Benjamin Disraeli “It is time for a new generation of leadership, to cope with a new problem and new opportunities. For there is a new world to be won.” JF Kennedy. In June 2007, the full genome of James Watson, codiscoverer of the structure of DNA in 1953, was resequenced, marking the gateway to an impending era of personalized genomic medicine. The importance of genomics to future medicine cannot be overemphasized, and so too are stem cells. As advocates for sick children, we must strive to provide our patients with the best possible treatment. Paediatric surgeons should therefore actively participate in the genomic and stem cell revolution to ensure that future paediatric surgery will be predictive, preventive, and personalized.
Acknowledgment I thank all my colleagues who have generously contributed to the work described, especially Drs VCH Lui, MM Garcia-Barcelo, E Ngan, Y Chen, KKY Wong, X Miao, and Ms MT So, and colleagues at the Genome Research Centre, the University of Hong Kong. The studies were supported by grants from the Research Grants Council
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Hong Kong and the University of Hong Kong. Many scientists and pediatric surgeons around the world have published relevant important papers that are not cited in this article because of space limitations.
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