Veterinary Oncological Pathology – Current and Future Perspectives

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The Veterinary Journal 2002, 163, 7±18 doi:10.1053/tvjl.2001.0637, available online at http://www.idealibrary.com on

Review Veterinary Oncological Pathology ± Current and Future Perspectives SUSAN M. RHIND Department of Veterinary Pathology, University of Edinburgh, Royal (Dick) School of Veterinary Studies, Easter Bush Veterinary Centre, Roslin, Midlothian, EH25 9RG, UK

SUMMARY Recent years have seen an explosion in the techniques available for detailed analysis of histopathological samples allowing improvements to be made in terms of both accuracy of diagnosis and, in certain instances, providing important prognostic information. The two broad areas where most interest has focused are in the investigation of cellular proteins/protein products by immunohistochemistry and the analysis of genes and transcripts using a range of molecular techniques. The numbers of reagents available for immunohistochemical applications in veterinary species are steadily increasing although still lag significantly behind the human diagnostic field in this respect. Molecular techniques currently in use include the polymerase chain reaction (PCR) and in situ hybridisation (ISH). More recent advances in terms of molecular analysis include the techniques of microarray, laser capture microdissection and proteomics which allow analysis of the genetic and protein repertoire of individual cell populations. This technology is extremely powerful with the potential to provide vast amounts of data. This review focuses on these techniques as they apply to the detailed analysis of # 2002 Harcourt Publishers Ltd tumours. KEYWORDS: Diagnostic pathology; immunohistochemistry; microarray; LCM. INTRODUCTION Routine pathological analysis of tissue samples (biopsy or post mortem) is based upon description of tissue structure and the morphological appearance of individual cells. This is generally carried out by light miscroscopy of routinely stained sections with the possibility of applying electron microscopy to appropriately fixed samples where ultrastructural examination is indicated. In recent years, it has been possible to extend this basic descriptive analysis to ask more specific questions about these cells which cannot be answered on a morphological basis alone. In particular, investigation of the expression of cell surface or intracellular markers by immunohistochemistry (IHC) to indicate the likely Correspondence to: Dr S. M. Rhind, Department of Veterinary Pathology, Royal (Dick) School of Veterinary Studies Easter Bush, Veterinary Centre, Roslin, Midlothian, EH25 9RG, UK. Fax: 0131 445 5770; E-mail: [email protected] 1090-0233/02/010007 ‡ 12 $35.00/0

tissue of origin has become an increasingly useful technique in oncology and is now available in most commercial diagnostic pathology laboratories. Further analysis of cells and tissues at the DNA/ RNA level is facilitated by the use of various molecular techniques which currently have their greatest impact in research and academic environments, however there are ever increasing examples of their application to diagnostics. A broad overview of the progression of these technologies is illustrated in Fig. 1, which illustrates the massive advances that have been made from basic morphological analysis of cells through to the situation we are now approaching where the entire genetic and protein repertoire of single cell populations can theoretically be assessed. Whenever embracing such new technology, however, there is inevitable conflict between methodologies which produce genuinely useful diagnostic/prognostic information versus what may be # 2002 Harcourt Publishers Ltd

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Analyse DNA/RNA

‘in situ’ Analyse y ‘in situ’

Fig. 1. Progression of technology in diagnostic pathology. LM ± light microscopy; EM ± electron microscopy; IHC ± immunohistochemistry; PCR ± polymerase chain reaction; ISH ± in situ hybridisation; FISH ± fluorescence in situ hybridisation, LCM ± laser capture microdissection.

considered as mere satisfaction of academic curiosity. The aim of this review is to provide an up to date overview of many of the technologies now available with discussion of their current (and likely future) relevance to veterinary oncological pathology. IMMUNOHISTOCHEMISTRY The technique of immunohistochemistry has been available for many years and involves the use of

specific monoclonal or polyclonal antibodies, which recognise defined antigens. In its simplest form, this technique allows identification of specific pathogens such as viruses, bacteria and protozoa ± these indications are well recognised and are outwith the scope of this review. Although present for many years in research environments and in human diagnostic fields, it is only relatively recently that immunohistochemical techniques have been applied with any regularity and consistency to animal samples for

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diagnostic purposes. The major constraint to the use of this technique has been the lack of reagents available. Since it is generally not commercially viable to generate specific reagents for use in domestic species, we often rely on `fortuitous' cross reactivity between human and animal antigens supplemented by reagents which become available as the result of species-specific research activities. Further limitations revolve around the fact that many antigens become `masked' during the fixation process, generally as a result of formalin induced cross linking of proteins. This can either render antigens redundant in terms of identification in anything other than frozen tissues or necessitate the use of an antigen unmasking technique such as microwaving or trypsinisation (Ezaki, 2000; Frost et al., 2000). The aim of utilising IHC in diagnostic veterinary oncological pathology is to provide an extra `layer' of information in addition to the standard morphological analysis. The major areas which will be discussed are phenotyping of lymphoid populations, analysis of the intermediate filament characteristics of cells, markers of metastasis and proliferation markers.

Lymphoid phenotyping

Specific antibodies allowing classification of subsets of lymphocytes have been used in research for many years. One major diagnostic application which is currently gaining momentum within veterinary diagnostic laboratories is in the classification of lymphosarcoma (especially canine) as being either of B cell or T cell origin. Antibodies against antigens specific to these cell types are available commercially and are applicable to routinely fixed and processed material. Antibodies in routine use in our laboratory include the T cell marker CD3 (Rabbit anti-human CD3, DAKO) and the B cell marker CD79a (Mouse anti-human CD79a, DAKO). One study has shown shorter remission and survival times for T-cell tumours compared to B cell tumours allowing useful prognostic information to be gained (Greenlee et al., 1990). Further markers are available for use on fresh frozen tissues or cytology preparations and include CD4 and CD8 (Moore et al., 1992) and CD21 (Moore, 1990). These markers were included in a panel used by Fournel-Fleury et al. (1997) to assess the similarities between canine lymphosarcoma and human non-Hodgkins lymphomas (NHL) using cytological analysis demonstrating notable differences between the two species. In addition to this application for defining the specific cell type involved, on a more basic level,

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lymphoid markers can also be used to establish lymphoid origin of poorly differentiated tumours or tumours which may be classed as being of `roundcell' origin with no clearly defined features. In the example in Fig. 2, an oral mass from a dog was diagnosed as a round cell tumour with a strong suspicion of malignant melanoma on the basis of light microscopic appearance. A panel of mAbs were applied to the mass including intermediate filament markers (see below) and lymphoid markers. The mass revealed diffuse CD3 positivity allowing diagnosis of epitheliotropic lymphoma and issuing of a different prognosis than would have been appropriate for the originally suspected malignant melanoma.

Intermediate filaments

Intermediate filaments are cellular skeletal proteins so called because they are intermediate in size between small actin filaments and large microtubules. Immunostaining for these proteins is a commonly used technique to elucidate the histogenesis of tumours where there is confusion based on the morphological appearance. Specifically, the presence of cytokeratin (CK) identifies cells of epithelial origin; vimentin, cells of mesenchymal origin; glial fibrillary acidic protein (GFAP) is present in glial cells; desmin in cells of muscle origin and neurofilaments in cells of neural origin. An example of the use of these markers is given in Fig. 3. In this example a poorly differentiated tumour was identified as a sarcoma (ultimately chondrosarcoma) despite many areas having a packeted `carcinomatous' appearance. In addition to these applications for biopsy specimens, there is obvious potential for application of this technique in diagnostic cytology. For example, panels of mAbs including intermediate filaments have been used in the differentiation of metastatic malignancies (Gupta et al., 2000). Despite the potential usefulness of this technique, the caveat remains that in situations where it may be most applicable, e.g. anaplastic tumours which fail to exhibit classical cytological differentiation, it may in fact be less useful given the propensity of some such tumours to lose differentiating features and protein expression. A further development in recent years in this field is the ability to predict on the basis of cytokeratin staining the likely primary source of epithelium in cases presenting with metastatic disease. The cytokeratin staining described above to differentiate epithelial from mesenchymal neoplasms generally uses a `broad-spectrum' mAb (the reagent in use in

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Fig. 2. (A) Histological section of an oral tumour from a dog showing infiltrate of round cells into the lamina propria and epithelium. Scattered melanin containing cells also present. Arrowhead ± epithelium; arrow ± lamina propria. Magnification 100. (B) Immunohistochemistry for CD3 showing infiltration of epithelium by many CD3 positive cells. Arrowhead ± epithelium; arrow ± lamina propria. Counterstain haematoxylin. Magnification 250.

our laboratory is Immustain broad spectrum mAb, CKKES). However the cytokeratins are a family of antigens (20‡) which can be expressed in different combinations in different types of epithelium. Expression of specific CKs can be identified using defined CK mAbs with narrow specificity compared to the `broad-spectrum' reagents described above. For example, CK7 and CK20 expression were analysed in a series of canine and feline tumours providing information potentially useful in helping to discriminate among carcinomas from different primary sites such that carcinomas presenting as

metastatic disease could be better defined (De Los Monteros et al., 1999). In addition to the intermediate filaments, there are a number of other markers available again to assist in diagnosis of generally poorly differentiated neoplasms. Other markers in relatively frequent use in our laboratory include S-100 (expressed in neuroectodermal tissue), Neuron-specific enolase (NSE) expressed in nervous tissue and neuroendocrine cells and chromogranin (expressed in neural tissues and secretory granules of endocrine cells). A recent study of endocrine tumours in the dog confirmed

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B

C

Fig. 3. Serial sections of nasal chondrosarcoma from a dog. (A) Haematoxylin and eosin; (B) Immunohistochemistry for cytokeratin; (C) Immunohistochemistry for vimentin. Almost all the cells are characterised by intracytoplasmic strong staining for vimentin. Counterstain haematoxylin. Magnification 400.

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the usefulness of this technique in identifying the presence of secretory granules in endocrine tumours (Doss et al., 1998).

Markers of metastasis

Adhesion molecules are cellular proteins involved in cellular adhesion and in some cases, in maintenance of tissue architecture. Logically, alterations in expression of these molecules may therefore be predicted to be associated with metastasis. This has been demonstrated for several tumours in humans including breast cancer in which reduced expression of the adhesion molecules E-cadherin or catenins has been shown to have an association with metastases and consequently a poorer prognosis (Yoshida et al., 2001). A study of canine mammary tumours also indicated reduced E-cadherin in mammary adenocarcinomas (Restucci et al., 1997).

Proliferation markers

A further application for immunohistochemistry which again can `bridge the gap' between research and diagnostic applications is in the analysis of the proliferative capacity of populations of cells. In contrast to the phenotyping described above which essentially further defines the static nature of a population, these markers allow an assessment of the dynamic capacity of the cell populations under investigation. The traditional method of assessing proliferation is by counting of mitotic figures however this can be inaccurate as a result of a number of variables including standardisation effects, reader subjectivity and error, variability in the mitotic phase and poor correlation with certain proliferation markers (van Diest et al., 1998). A range of other techniques exist to assess the proliferative nature of neoplastic masses and many have been applied to animal tumours. These techniques can be broadly categorised into immunohistochemical techniques, incorporation techniques and analysis of Nucleolar Organiser Regions (NORs). The broad principles of these techniques are illustrated in Fig. 4. Immunohistochemistry. The two most common proliferation associated antigens which have been investigated by this technique are the proliferating cell nuclear antigen (PCNA) and the Ki67 antigen. PCNA is a nuclear protein which has a critical role in DNA synthesis; as such it is maximal in S-phase cells. Ki67 is a nuclear antigen present in all stages of the cell cycle except G0 and early G1.

Several studies have investigated PCNA expression in canine tumours with variable results. Useful prognostic information was indicated in mast cell tumours (Simoes et al., 1994) whereas it was less useful in melanocytic tumours (Roels et al., 1999) and lymphoma (Kiupel et al., 1998; 1999). The Ki67 antigen is best demonstrated by the mAb MIB-1 in fixed tissue and is generally regarded as a more reliable marker of proliferating cells (van Diest et al., 1998). This marker has been successfully used to demonstrate the proliferative capacity of tumours in domestic species including melanocytic tumours (Roels et al., 1999), plasmacytic tumours (Platz et al., 1999), lymphoma (Kiupel et al., 1999; Fournel-Fleury et al., 1997) and mast cell tumours (Abadie et al., 1999). As a prognostic indicator, it has also proved reliable in several human tumours including lymphomas (Yamanaka et al., 1992). Incorporation techniques. The major incorporation technique which has been used is that of Bromodeoxyuridine (Brdu) incorporation. Brdu is a thymidine analogue and is incorporated into cells in S-phase of the cell cycle (Fig. 4B). This technique requires injection of Brdu in vivo prior to surgical removal of the tissue under investigation and subsequent identification of incorporated Brdu by immunohistochemistry. The obvious drawback of this technique is the requirement for administration of the compound to the whole animal and hence by definition is a more invasive technique than standard IHC. The advantage is that this technique has been considered as a `gold-standard' in terms of identifying cells in S phase (van Diest et al., 1998). A study using this technique in oral tumours in dogs indicated it as a potentially useful technique for evaluating prognosis (Yoshida et al., 1999). AgNORs. A further technique worthy of mention in the context of proliferation markers is that of analysis of nucleolar organiser regions (NORs). The principle of this technique is illustrated in Fig. 4D. These are argyrophylic (Ag) and hence this analysis is often referred to as AgNOR assessment. The potential benefit of this technique over those described above is that it allows comment on the magnitude of the proliferative response rather than a simple `on±off' signal given by immunohistochemistry for proliferation markers such as PCNA or Ki67 (Fig. 4A). Again this technique has been used to provide prognostic information in a range of canine tumours including mast cell tumours (Simoes et al., 1994). A further recent study describes a technique

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Brdu Incorporation

Ki67, PCNA, Brdu

Fig. 4. Markers for assessing proliferation in cell populations. (A), (B) ± cell cycle illustrating principal of Ki67, PCNA and Brdu analysis. (C) ± qualitative information provided by staining for these markers. (D) ± potentially quantitative information obtained by AgNOR staining.

for optimisation of AgNOR and PCNA quantification in a range of canine tumours again demonstrating their use in providing objective measurements useful in differentiating benign and malignant tumours (Hung et al., 2000). MOLECULAR TECHNIQUES ± THE PRESENT AND THE FUTURE The standard molecular techniques currently in use in veterinary diagnostic pathology include the polymerase chain reaction (PCR) and in situ hybridisation (ISH). As is well recognised, PCR allows the amplification of specific DNA sequences from tissue samples whereas in situ hybridisation provides a further layer of information in that specific

sequences are detected by either DNA or RNA probes allowing precise cellular localisation of target sequences. Currently, these techniques are of greatest use in identification of infectious agents as frequently described in the literature (Alleman, 1996; Brown, 1998). With specific relevance to oncological pathology however, modern molecular techniques involve the identification of oncogenes or specific DNA alterations which can be detected by relatively simple PCRbased and hybridisation methodologies (reviewed by Naber, 1994). A more recent advance on the technique of PCR is that of `Real-time' PCR. This is essentially a kinetic technique which allows for an element of gene quantitation which is not generally reliable with standard PCR technologies as a result of

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the number of variables in the reaction. As for `standard' PCR and in situ hybridisation techniques, this technology has greatest impact in terms of rapid identification of pathogens; however applications in oncology are also frequently reported. For example, Peter et al. (2001) describe the application of this technique to small round cell tumours of children where detection of gene fusions specific for different tumours can be detected simultaneously. A further interesting example describes the use of this technique for the detection of tumour cells in the blood of patients with hepatocellular carcinoma (Miyamoto et al., 2001).

Microarrays (MA) and laser capture microdissection (LCM)

These technologies are relatively novel and in the human field are currently more applicable to research than genuine diagnostic applications. Nevertheless, the technology is inexorably moving into the diagnostic field with ever increasing

examples of cases where useful information has been gained. Both technologies will initially be dealt with separately although in terms of applications for diagnostic pathology, they can be used sequentially as will be described below. Microarrays. The principle of microarray technology is relatively simple and is illustrated in Fig. 5. In this example, the procedure involves `arraying' of many (often thousands) of DNA sequences (either short oligonucleotides or longer cloned fragments of DNA) onto glass slides or membranes. The slide is then `interrogated' by two samples ± one from a control population, the other the sample under test. Both samples are labelled with a different fluorescent probe and they competitively react with the arrayed cDNA molecules. Following incubation, unbound probe is washed off and slides are analysed for binding of none, one or both samples to the target. By way of example, this technology has recently been applied to the analysis of diffuse large

Fig. 5. Principle of microarray. Multiple DNA `spots' arrayed onto slide. Labelled DNA samples applied and binding analysed.

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B-cell lymphoma in humans (Alizadeh et al., 2000). The authors designed a microarray which they called a `Lymphochip' containing selectively chosen genes suspected to be involved in the genesis of these tumours with the aim of defining specific `signatures' for individual tumour subtypes. Using this technique, they identified two molecularly distinct forms of diffuse large B-cell lymphoma associated with significantly different survival rates. The potential amount of data which can be obtained using such technologies is vast and it is important to consider whether in reality this technique is effectively only a research tool with little potential for application in the diagnostic arena. A recent review by Snijders et al. (2000) discusses this issue, identifying the potential uses in human pathology for the technique including tumour classification, biological staging of tumours and detection of microorganisms. However the practical statement is made that even in human pathology `there are still more people discussing the technology than using it'. Generation of the quantity of data emanating from microarray technology will require massive bioinformatics support in terms of analysis

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of the significance of the results. This is potentially problematic enough in the human field even with the prospect of the Human Genome Project reaching its conclusion in the near future. However one further drawback of generating this type of data in veterinary species is the relative lack of baseline sequence data currently available. For these reasons, at least at present it would appear sensible to proceed with caution in this area since it is difficult to predict when, if ever, this technology will become applicable to veterinary pathology. Laser capture microdissection. This relatively new technique (first described by Emmert-Buck et al. (1996)) adopts the simple principle of using microdissection to remove specific cells of interest from a tissue section on a glass slide (illustrated in Fig. 6.). The major advantage of the technique is that genetic information from individual cell populations can be analysed and compared to adjacent `normal' cells thus overcoming the problem of tissue heterogeneity. One fascinating application of this technique revolves around the ability to select for example normal, neoplastic and metastatic cell populations and

Fig. 6. Principle of Laser Capture Microdissection. 1 ± laser beam passed through plastic cap onto to cells of interest. 2. Cells of interest removed by adhesion to transfer polymer. 3. RNA/DNA isolated from cell population for further analysis.

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analyse differences in the genetic `make-up' of these individual cells. Clearly this particular example is more of a research issue than having direct clinical relevance, however, there are increasing examples of the application of this type of work in human medicine. One published case example describes shared molecular abnormalities in tumour tissue from a patient with a previously resected intestinal tumour. This analysis revealed a common clonal origin in tumour tissue from two other sites despite differing histological and immunophenotypic characteristics. This established a definitive diagnosis of tumour recurrence and metastasis of tissue from the three sites (Milchgrub et al., 2000). It can be predicted with a fair degree of certainty that the numbers of such cases reported will rise rapidly as more cases are analysed in this way. Ultimately, it is likely that many tumour specific genetic profiles will be established with implications for both diagnosis and prognosis. The technique of LCM has been further extended by combining it with immunohistochemistry which may have indications in cases where morphological features alone are not sufficient to identify the cells under investigation (Fend et al., 1999). Potentially the most powerful application of this technology is the combining of LCM with microarray leading to the ability to analyse the complete genetic repertoire of individual cell populations (Fig. 1). This could be considered to represent the ultimate in diagnostics with the ability to analyse at the highest resolution individual cellular genetic abnormalities. However, despite the overall attractiveness of such a situation as alluded to above, the financial input required to achieve this data and its usefulness given the relative lack of genetic knowledge in veterinary species currently renders it an impractical technique to be used in any routine sense.

Cytogenetics

The field of cytogenetics (i.e. chromosomal analysis) is vast and as such a complete review of this topic is outwith the scope of this article, nevertheless a brief summary of current applications is relevant given its role and importance in the molecular diagnosis of certain cancers. In a similar way to standard in situ hybridisation, analysis of chromosomal abnormalities in situ can be carried out using the technique of Fluorescent in situ hybridisation (FISH). This technique retains histological architecture whilst allowing for detection of aberrant chromosomes in situ. As more information is learned about chromosomal abnormalities associated with specific forms of cancer, labelled DNA

probes can be manufactured to allow detection of cells with features consistent with malignancy. A related technique is that of comparative genomic hybridisation (CGH) in which evaluation of chromosomal material can be carried out from both fresh and archival material. In simple terms, this technique compares normal DNA with that derived from tumour tissue and highlights differences (gains or losses of DNA across the genome) between the two (James, 1999). This technique has been used extensively in humans ± especially in analysis of breast cancer (e.g. Gunther et al., 2001) and other cancers including that of the prostate (Chu et al., 2001). The first application of the technique to the dog has recently been described by Dunn et al. (2000), who developed a technique for CGH to analyse DNA from a glial tumour cell line.

The post genomic era: proteomics

The global analysis of cellular proteins is called proteomics whereby various techniques are used to resolve and characterise proteins (Blackstock & Weir, 1999). Whilst the techniques described above are essentially modifications of, and more technical mechanisms for analysing gene expression profiles, it is becoming apparent that the study of proteins in a similar `high resolution' way may in fact provide more meaningful information. Reasons for this include discrepancies between mRNA levels and active protein levels and the fact that gene analysis does not address the issue of post-translational modification of proteins (Chambers et al., 2000). Briefly, this technique involves solubilisation and electrophoretic separation of proteins followed by mass spectrometry to identify proteins of interest. The current most popular system for this analysis is matrix-assisted laser desorption time-of-flight mass spectrometry, commonly referred to as MALDI-TOF. Despite the potential power of this technique however a major current drawback with regard to diagnostic pathology applications is the requirement for fresh tissue as formalin fixation renders the constituent proteins insoluble (Chambers et al., 2000). The potential for this technique with regard to oncology, is in the generation of `proteome profiles' for particular tumours as are currently being established for example for human uroligical malignancies (Unwin et al., 1999). CONCLUSIONS Whilst the indications and applications for the use of immunohistochemical techniques are clear with

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many examples of genuine indications for their use available throughout the literature, it could be considered that the `jury is still out' in terms of the genuine practical applications for the more advanced molecular techniques described above. Although it could be argued that in many situations, this `downstream' analysis of cellular phenotype is not providing crucial prognostic information, and is thus more of an exercise in academic satisfaction, there are increasing instances of prognostically useful information being forthcoming from these techniques. The number of publications addressing this topic and the general interest in the scientific community in this `molecular revolution' will ensure that this area continues to be high profile. Furthermore, although the benefits of applying these techniques may not be initially apparent, it is only by constant enquiry and aiming for definitive answers that novel disease phenotypes/patterns can be established. Accepting this however, there is nevertheless an appreciation of the financial repercussions of utilising these techniques such that inevitably in the commercial world, such techniques will be reserved for cases in which genuinely useful prognostic information can be gained. In conclusion, it is vital to appreciate that modern technologies can be an important and exciting adjunct to routine histopathological examination however the importance of the latter can never be overestimated. As stated by Jones and Fletcher (1999) we need to accept the `historical supremacy of routine histology' in diagnostic pathology. Nevertheless, we have progressed through phases of utilising markers to recognise specific features of cells both in terms of their phenotype and proliferative capacity ultimately to the capacity to individually dissect the individual genetic repertoire of specific cells and cell populations. It is important however that we continue to maintain realism in our application (and interpretation) of modern techniques as they become available. This will ensure that the delicate balance between enthusiasm for new technology and pragmatism can be maintained to the optimum benefit of our patients. ACKNOWLEDGEMENTS The author is grateful for the technical support of Neil MacIntyre, Sharon Moss and Andrew Dawson and to Cheryl Scudamore for critical review of the manuscript.

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