Nasopharyngeal Carcinoma

C H A P T E R

1 Nasopharyngeal Carcinoma: A History A.B. Rickinson1, K.W. Lo2 1

Institute of Immunology and Immunotherapy, Division of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom; 2Department of Anatomical and Cellular Pathology and State Key Laboratory of Translational Oncology, Chinese University of Hong Kong, Hong Kong, China

O U T L I N E Nasopharyngeal Carcinoma: An Introduction

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Recognition of the Tumor and Its Unique Epidemiology

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Environmental and Lifestyle Factors

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Determinants of Genetic Susceptibility

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The Link to EpsteineBarr Virus

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The Impact of Tumor Genomics

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Toward an Understanding of Nasopharyngeal Carcinoma Pathogenesis

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Development of Nasopharyngeal Carcinoma Therapies

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Nasopharyngeal Carcinoma: A Future Perspective

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References

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NASOPHARYNGEAL CARCINOMA: AN INTRODUCTION Nasopharyngeal carcinoma (NPC) of undifferentiated type is a tumor whose unique pathogenesis, involving genetic, lifestyle, and viral cofactors, has made it an important platform for research on the multifactorial nature of oncogenesis. Here we follow the various strands of that research as they have grown over the past 50 years, diverging and reconnecting with one another to furnish our current understanding of NPC development. We describe the emergence over the same period of radiotherapy and chemoradiotherapy as standard treatment options, then consider how fundamental research is raising the prospect of novel therapies and even the possibility of tumor prevention. In each of these areas we highlight what we see as the most important findings to date, identify the challenges that remain, and, with an eye to the lessons of the past, discuss how best to meet those challenges in the future. NPC arises from epithelial cells within the lymphocyte-rich nasopharyngeal mucosa, with an epicenter in the Fossa of Rosenmuller, a pharyngeal recess within the nasal cavity from which the tumor has multiple routes of spread. In particular, this location helps to explain why NPC so frequently presents as a lymph node metastasis in the neck rather than as a primary nasal lesion. Histologically the tumor is characterized by malignant epithelial cells that are of squamous origin but lack keratinization and are classified as either undifferentiated or poorly differentiated [Fig. 1.1]. Tumors with either of these two closely related histologies, originally designated NPC World Health Organization (WHO) types 2 and 3, are now considered as a single clinical entity, undifferentiated (nonkeratinizing) NPC.1 One characteristic feature of this tumor’s histology is the presence of a rich lymphocytic infiltrate whose content and biological significance is still poorly understood. Note that there is also a well-differentiated (keratinizing) nasopharyngeal tumor, originally designated NPC WHO type 1, which is not only histologically distinct from the undifferentiated carcinoma but has a quite different pathogenesis and epidemiology. Our focus here is on the undifferentiated (nonkeratinizing) form of NPC. Nasopharyngeal Carcinoma https://doi.org/10.1016/B978-0-12-814936-2.00001-8

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Copyright © 2019 Elsevier Inc. All rights reserved.

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1. NASOPHARYNGEAL CARCINOMA: A HISTORY

(A)

(B)

X400

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FIGURE 1.1 Nasopharyngeal nonkeratinizing carcinoma. (A) Undifferentiated subtype. The syncytial sheets and scattered carcinoma cells exhibit vesicular nuclei, prominent nucleoli, scanty pale esinophilic cytoplasm without clearly discernible cytoplasmic borders and admixed with dense lymphoplasmacytic infiltrate. (B) Poorly differentiated carcinoma. The sheets of carcinoma cells have a squamoid appearance, exhibit more abundant amounts of esinophilic cytoplasm with well-defined cytoplasmic borders. Focal suggestion of intercellular bridges is noted, but keratinization is not seen.

RECOGNITION OF THE TUMOR AND ITS UNIQUE EPIDEMIOLOGY The tumor’s frequent presentation in metastatic form, as a swelling in the neck with superficial resemblance to an inflammatory tuberculous lesion, no doubt contributed to the lateness of its recognition as a malignancy of nasopharyngeal origin. As reviewed by van Hasselt and Gibb,2 the first clinical description of an NPC in the Western literature has been attributed to Durand-Fardel in 1837.3 However it was not until the early years of the 20th century that the nasopharyngeal origin of the tumor was first recognized, and NPC acquired textbook status as a rare but distinct entity.4 Somewhat surprisingly, early Chinese medical records do not specifically mention the tumor, perhaps reflecting both the difficulty of clinical diagnosis and the fact that such records were largely drawn from central and northern regions of China where the disease was less prevalent than in the south. In 1921, Todd, working in Canton (now known as Guandong), was the first to describe a series of cases in Chinese patients,5 followed in 1930 by a comprehensive report from Digby and colleagues of the tumor’s clinical, anatomical, and histological features based on >100 cases in Hong Kong.6 For many years the cellular origin of NPC remained in doubt and different interpretations of the tumor’s histology led to its description as an endothelioma, a reticulo-endothelioma, or a carcinosarcoma. Subsequently the term “lymphoepithelioma” gained currency, reflecting the mixed cellular composition of the tumor mass and the uncertainty over which were the malignant cells. Indeed it was not until the first WHO classification in 1978 that a consensus was reached and the epithelial origin of the tumor was formally recognized.7 By then, interest in NPC had grown considerably because of its distinct epidemiology. First, it had an unusual age profile, occurring throughout adult life but with a marked incidence peak in 45e60 year-olds, and a male predominance (M:F ratio 3:1). Second, and more important, incidence rates varied hugely between different ethnic groups, with Southeast Asian people particularly at risk. A recent International Agency for Research on Cancer (IARC) Globoscan profile of worldwide NPC incidence is shown in Fig. 1.2, although the homogenization of data from any one country tends to obscure important detail; for example, the very strong gradient of increased incidence as one moves from north to south within China. Already by the 1960s,8 many studies had reported the high incidence of NPC (agestandardized rate in males of >15 per 100,000) among Southern Chinese, with an epicenter around Canton that extended down the Pearl River delta to include Hong Kong. Such rates are at least 30-fold greater than those seen in Caucasian populations. Migrants of Cantonese descent in Singapore have similarly high rates, as do firstgeneration Cantonese migrants to California. The tumor is also seen more generally throughout Southeast Asian populations, typically at intermediate incidence rates (age-standardized rate in males >4 per 100,000), but with hotspots of high incidence in certain minority ethnic groups in island nations of the Southeast Asian archipelago. Interestingly Wee and colleagues9 have proposed that such groups, like Cantonese people themselves, are descendants of a high-risk aboriginal people who, at the end of the last Ice Age some 10,000 years ago, lived in the area of the South China Sea and were displaced in multiple directions by rising sea levels. The relevance of this hypothesis to foci of

ENVIRONMENTAL AND LIFESTYLE FACTORS

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FIGURE 1.2 Timeline showing the major milestones in nasopharyngeal carcinoma (NPC) research. Above the timeline are shown the major findings in NPC xenograft/cell line establishment (top row), EpsteineBarr virus (EBV)/cell interactions (middle rows) and EBV-related diagnostics (bottom row). Below the timeline are shown the major findings in NPC genomics (top row), environmental cofactors (second row), genetic predisposition (third row), and therapies (bottom row).

high NPC incidence beyond Southeast Asia remains conjectural. Interestingly, intermediate to high rates of NPC are seen in Inuit populations,10 themselves of Asian descent, in Arctic regions, but NPC is also quite common in countries of the North African Maghreb and, reportedly, in certain communities in East Africa.8 To what extent these different incidence rates reflected an inherent genetic susceptibility versus the influence of local environmental factors became a key issue. Almost all the relevant work on this question has involved people of southern Chinese ethnicity. In this regard, the epidemiologic studies of Buell in the 1960s and 1970s11,12 were of seminal importance. Using medical records stretching back almost 100 years, he examined NPC incidence in the Chinese (largely Cantonese) diaspora in California. He showed that, while first-generation (Chinese-born) immigrants had the same high risk as the population of Canton, NPC incidence in their (US-born) descendants fell by at least twofold in the second generation and by an additional twofold in the third. This implied that at least part of the high risk among native Cantonese people was associated with some environmental/lifestyle factor to which US-born immigrants were progressively less exposed. This was not the whole story, however, since this third generation was still at 10-fold higher risk of NPC than the local Caucasian population, strongly implying that genetic susceptibility was another factor. From this point on, we see two separate strands of research moving forward, one focusing on the environmental/lifestyle factors that might increase tumor risk, the other seeking to identify the determinants of genetic susceptibility. These can be traced as part of a wider timeline of NPC research shown in Fig. 1.3.

ENVIRONMENTAL AND LIFESTYLE FACTORS The fact that Chinese-born immigrants, typically arriving in California as young adults, carried the same high risk of NPC as the Chinese population they had left behind suggested that the predisposing effects of environmental/ lifestyle factors were mediated in infancy or childhood. Buell also reached a similar conclusion by contrasting the age profiles of NPC and another epidemiologically well-studied tumor, lung cancer.11 The latter, which we now know is largely caused by long-term cumulative exposure to carcinogens in cigarette smoke, shows an exponentially increasing incidence throughout adult life, rates rising in proportion to the seventh power of age. NPC’s age profile is quite different, increasing in proportion to only the second power of age, a relationship inconsistent with chronic cumulative exposure and implying a limited period of environmental exposure occurring early in life. The question of what that exposure might be has long been of interest, with the origin of the tumor in the Fossa of Rosenmuller implying a role for a carcinogen entering the airways either from the atmosphere or from volatile components in the diet. In that regard Peter Clifford, an ENT surgeon working in Kenya in the 1960s, first linked an

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1. NASOPHARYNGEAL CARCINOMA: A HISTORY

FIGURE 1.3 Global distribution of nasopharyngeal carcinoma incidence rates. Age-standardized rates (ASR) in males (all ages) per 100,000 person/year. The cities/regions/populations with high incidence of NPC are indicated. The map was drawn on the basis of GLOBOCAN 2012 and Cancer Incidence in Five Continents XI, IARC.

increased incidence of NPC in one tribal group, the Nandi, to their traditional lifestyle with long periods in smokefilled huts.13 However to John Ho, a radiologist/oncologist and the pioneer of NPC studies in Hong Kong, smoke exposure could not explain the high rate of NPC in Cantonese people. Having been struck by the number of his patients coming from the “boat people” of Hong Kong harbor, Ho showed that NPC incidence was indeed doubled in that community compared to the general population.14 He inferred that some environmental/lifestyle factor common to many Cantonese was exaggerated in the boat peoples’ traditional lifestyle, and this could not be smoke exposure since these people cooked and ate on deck in the open air. In a very influential and wide-ranging review of NPC,15 he proposed that the environmental/lifestyle factor was the widespread practice of weaning infants onto a diet of Cantonese-style salted fish, made from the whole contents of the fish without gutting, and sometimes left to soften by decomposition before salting in nitrate-containing preservatives. Ho’s ideas have since been tested in a series of retrospective epidemiological studies comparing the early lifestyles of NPC patients and population controls in Hong Kong, mainland China, Taiwan, and Malaysia. Remarkably, all showed that a history of eating salted fish early in life was significantly linked to NPC risk.16e21 While some studies raised the possibility of other dietary or occupational exposures acting as additional independent risk factors, the link to salted fish remained a constant. Mechanistic support has since strengthened the argument, showing that traditional Cantonese salted fish contains a series of volatile nitrosamines, chemicals with known oncogenic potential.22 Indeed such findings stimulated a parallel search for similar carcinogens in the diets of other intermediate/ high NPC risk populations, namely the preserved fish eaten by Inuit people and in traditional Tunisian spices.22 However, the smaller numbers of patients in these other areas have precluded the kind of robust epidemiological studies that have lent their weight to Ho’s salted fish hypothesis in the Cantonese context.

DETERMINANTS OF GENETIC SUSCEPTIBILITY The early epidemiological studies of Buell had strongly suggested that genetic susceptibility was at least as important as lifestyle in determining the unusually high incidence of NPC in people of Southern Chinese descent. This was further supported by a series of anecdotal reports, first appearing in the 1970s, of familially clustered cases of the disease. Such examples were seen both in high and low NPC incidence populations,23,24 and indeed it is now

DETERMINANTS OF GENETIC SUSCEPTIBILITY

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estimated that 10% of all NPCs involve individuals from families where one or more members have a history of the disease.25 First-degree relatives of NPC patients clearly have a greater risk of developing the tumor, with a median value for increased risk across different reports of around fivefold. There are no obvious clinical, or as yet biological, differences between familial and sporadic NPC, except that familial cases tend to be disproportionately represented among the small subset of tumors categorized as “early onset” (patients <40 years age); accordingly, first-degree relatives of early onset cases have an even higher (around 10-fold) increased risk of developing the tumor. The first steps to unraveling the basis of such genetic susceptibility were taken in the mid-1970s through a collaboration between Malcolm Simons, an immunogeneticist at the WHO Immunology Research and Training Center in Singapore, and Guy de The, an epidemiologist with a specific interest in virus-associated cancers working at the IARC laboratories in Lyon. Although the field of human genomics was in its infancy at the time, immunologists interested in transplantation were developing tools to study the most polymorphic region in the genome, the histocompatibility leukocyte antigen (HLA) locus. Using the crude serologic assays then available for HLA typing, Simons and colleagues noted differences in HLA allele distribution between an NPC patient cohort, mainly Singaporeans of Cantonese descent, and ethnically matched healthy controls. Both the HLA-A2 allele (as then defined) and a blank HLA-B locus (i.e., an allele not defined by the Caucasian typing sera) were significantly more frequent in patients than controls.26e28 In one of the chance coincidences that abound in the history of science, this result was published within a few weeks of Zinkernagel and Doherty’s seminal discovery of major histocompatibility complex (MHC) restriction, that is the role played by MHC class I (in humans, HLA-A/B/C) molecules in mediating target cell recognition by CD8þ T cells.29 Seen in this light, the coincident findings of Simons and colleagues gained particular significance. Indeed they mark the starting gun for four decades of research into the link between NPC susceptibility and genomic polymorphisms, first at the HLA locus and subsequently across the entire genome. Numerous studies over that time, including a first analysis of affected individuals within NPC families,30 have indeed confirmed that the HLA region on chromosome 6p21 houses the strongest determinants of genetic susceptibility to NPC.30e33 The original findings of Simons et al. were confirmed as allele-specific DNA probes showed that an HLA-A, B haplotype unique to Southeast Asian people, HLA-A*0207, Bw46, was more frequently seen in NPC patients than healthy controls. Further studies have shown that other Southeast Asian alleles, namely A*0206 and the A*3301, B*5801 haplotype, are also associated with increased NPC risk. Even stronger is the effect of the HLA-A*11.01 allele, which has worldwide distribution but is surprisingly common in Cantonese people. Possession of this allele is clearly associated with a reduced NPC risk, with around 40% NPC patients being A*11.01-positive versus 50%e55% in controls. A link between HLA type and NPC risk has also been seen in studies of NPC patients and controls in both Inuit and North African populations.10,34 Though these studies are much smaller than in Southeast Asia and their conclusions preliminary, they make the important point that the candidate HLA risk alleles identified in those patient cohorts are quite different from those identified in Southern Chinese. The phenomenon of HLA-related NPC risk may therefore be a general feature of intermediate to high risk populations, but involve different susceptibility alleles. It is worth stressing that the HLA class I locus, which most studies on high risk Southern Chinese cohorts have identified as a critical region, actually spans >2 MB of DNA and contains >200 genes held together at high levels of linkage disequilibrium. A central issue therefore became whether the HLA genes themselves were the riskdetermining loci or simply markers that cosegregate with the actual key determinants as part of an extended haplotype. The onset of genome-wide association studies of single nucleotide polymorphisms has allowed this issue to be addressed in an unbiased way and, to date, the results suggest that the HLA alleles themselves are indeed the source of disease risk.33 Furthermore, the strongest associations with risk map to polymorphisms encoding residues in the HLA class I molecule’s peptide-binding groove (i.e., the domain that determines which peptide fragments of foreign antigens are presented to the T cell system). The implications of these findings for a virus-associated malignancy such as NPC will be discussed later in this chapter. As genomic analysis has become ever more sophisticated, the search for determinants of NPC susceptibility has extended across the entire genome. Again these studies have had a Southern Chinese focus and have identified several candidate genes, though none with the same degree of association to NPC risk seen in the HLA class I region. Significant links have been reported with single nucleotide polymorphisms in the MECOM gene region on chromosome 3q22 encoding the MDS1/EVI1 transcriptional regulator, in the TNFSFR19 gene on chromosome 13q12 encoding a receptor of the tumor necrosis factor (TNF) superfamily, in the CDKN2A/B region on chromosome 9p21 encoding a cyclin-dependent kinase inhibitor, and in the TERT region on chromosome 5p15 encoding telomerase.35,36 In parallel, whole exome sequencing studies focusing on early onset/familial NPC cases has reported a variant of the MST1R (macrophage stimulating 1 receptor) gene on chromosome 3p21 strongly associated with early

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1. NASOPHARYNGEAL CARCINOMA: A HISTORY

onset disease.37 However, even combining the preceding loci with HLA genes, the full extent of familial risk has still not been completely explained, and so the search for determinants of NPC susceptibility, and for the biological basis of their effects, continues to this day.

THE LINK TO EPSTEINeBARR VIRUS A third major strand of NPC research, running in parallel to studies of lifestyle and genetic cofactors, opened in 1966 with a completely unexpected finding, a possible connection between the tumor and a human herpesvirus, the EpsteineBarr virus (EBV). This virus had been discovered only 2 years earlier by Tony Epstein, Yvonne Barr and Bert Achong at the Middlesex Hospital in London.38 They were working on a quite different human tumor, but one with an equally fascinating epidemiology. Burkitt lymphoma (BL), as it came to be known, occurred at very high incidence among children in equatorial Africa but at the time had not been described elsewhere. However the virus that Epstein and colleagues discovered in cultured BL cells was not, as had been anticipated, one confined to equatorial regions of Africa but a herpesvirus that turned out to be a common infection in most populations worldwide. Such ubiquity fuelled serious doubts about the significance of EBV’s link to BL, and several laboratories had begun to address this issue by comparing virus-specific antibody levels in BL patients with those in controls. One of those laboratories, headed by Lloyd Old at the Sloan-Kettering Institute in New York, obtained BL sera for their studies from Peter Clifford in Kenya and, among his controls, Clifford serendipitously included sera from local NPC patients. Remarkably, Old and colleagues found elevated antibody titres against their BL-associated antigen (i.e., an extract of a virus-producing BL cell line) both in BL and also in NPC sera, but not in other controls.39 Independently Werner and Gertrude Henle in Philadelphia were developing an immunofluorescence assay specific for the EB virus capsid antigen (VCA) complex and in 1970, working with John Ho in Hong Kong, unequivocally showed that anti-VCA antibody titres were indeed elevated in Chinese NPC patients.40 Against all expectations, the EBV/ NPC link appeared to be real and universal. Serologic evidence had its limitations, however, and the crucial molecular evidence was yet to come. The detection of EBV DNA in extracts of NPC biopsies was first reported in 197041 but it was not for another 3e4 years that the critical issue, the actual cellular origin of that signal, was resolved. This was through the work of two laboratories, led by Harald zur Hausen in Freiburg and George Klein at the Karolinska Institute in Stockholm. Zur Hausen used in situ hybridization with virus-specific probes to detect EBV DNA in the malignant epithelial cells and not in cells of the lymphoid infiltrate.42 Meanwhile Klein applied his recently developed immunofluorescence test for the EBV nuclear antigen (EBNA) complex to the first NPC-derived nude mouse-passaged cell line, made by Beppino Giovanella in Houston, and showed that the epithelial tumor cells, now passaged free of any lymphoid infiltrate, were uniformly EBNA-positive.43 Through such key findings, the concept of EBV as an etiologic factor in NPC’s development now gained a firm foothold; indeed, among the increasingly wide spectrum of EBV-associated tumors, NPC is one of those with the highest disease burden in terms of world health and one of only two (the other being a rare nasal T/NK lymphoma) that is consistently EBV-positive irrespective of population ethnicity and incidence rates.44 Over the ensuing four decades, research on the EBV and NPC has made substantial progress in several areas (see Fig. 1.3), each taking advantage of the virus/tumor connection and addressing issues that are of long-term significance. Here we describe progress in two of the most important areas, first on understanding the virus’s fundamental role in the oncogenic process and second on the use of viral markers as invaluable tools in NPC diagnosis; a third area of importance, how EBV’s involvement is opening up new possibilities for treatment and even prevention of the tumor, will be discussed later. Mechanisms of viral oncogenesis: Establishing the link between EBV and NPC was especially surprising because, at the time, EBV was known as a strictly B lymphotropic agent, indeed one with potent B cell growth-transforming ability in vitro,45 and there was no prior evidence of it infecting epithelial cells. How the virus might behave in an epithelial environment was unknown, particularly as epithelial cells were refractory to conventional EBV infection in vitro. Following the publication of the EBV genome sequence in 1984,46 studies of EBV-transformed B lymphoblastoid cell lines (LCLs) soon identified the six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and eLP) and two latent membrane proteins (LMPs 1 and 2) whose constitutive expression, along with the ubiquitous EBER1 and EBER2 RNAs, defined the transformed B cell state. By contrast, however, viral protein expression in freshly established Burkitt cell lines was found to be restricted to a single protein, EBNA1.47 Thus the concept of alternative latencies was established, with BL categorized as Latency I. Rapid extension of the work to NPC then identified yet another form of restricted latency in this epithelial tumor, characterized by EBNA1 expression with some 25%e40% of tumors also detectably LMP1-positive48,49; to this was later added expression of LMP2 in most if not all

THE LINK TO EPSTEINeBARR VIRUS

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cases, so defining a Latency II infection. This pattern was not only confirmed by analyzing the virus-coded RNAs in NPC cells but, more importantly, was extended through the detection of abundant highly spliced rightward transcripts from the Bam HIA region of the genome (BARTs).50 The true significance of that finding was only realized 15 years later with the discovery of EBV-coded microRNAs (miRs), many of which were derived from BART intronic sequences51; though initially detected in a virus-transformed B cell line, such miRs were later shown to be expressed at unusually high levels in NPC.52,53 Understanding the role that these EBV latent proteins and noncoding RNAs play in NPC pathogenesis became a major line of research for many labs in the field, with studies in epithelial cell models running alongside parallel work in B cells. Most progress to date has stemmed from work on the LMPs. Both proteins function as constitutively activated receptors that link to key signaling pathways.54,55 Most importantly, LMP1 interacts with, among others, mediators of TNF receptor family signaling and thereby drives activation of the NF-kB pathway, including formation of the p50/p50/bcl3 and p50/relB heterodimers that were later found to be the dominant species in NPC cells; this has multiple effects favoring both epithelial cell growth and survival while blocking terminal differentiation.56 LMP2 in its full length form (LMP2A) also has multiple connections, of which its linkage to Akt activation is perhaps most relevant to epithelial malignancy through differentiation blockade and induction of a migratory phenotype.56 The effects attributable to the EBV-encoded miRs still remain to be elucidated but their importance, possibly as mediators of immune evasion and/or regulators of the tumor environment, is highlighted by the recent observation that in vivo passage of an in vitro-established NPC cell line is associated with marked amplification of BART miRNA expression levels.57 NPC’s unique epidemiology has also raised the intriguing possibility that certain EBV strains, notably strains prevalent in Southeast Asian populations, have increased carcinogenic potential. It became clear that, while all EBV isolates are highly homologous (98%e99%), there are individual sequence polymorphisms that act as signatures of strain identity, which lie mainly within the latent growth-transforming genes.58 Moreover, many of these polymorphisms act as geographic markers, distinguishing strains prevalent, for example, in Southeast Asian populations from those prevalent in Caucasians. Such geographic differences are not surprising given the thousands of years available for coevolution between virus and isolated host populations following human migration out of Africa, plus the potential influence of founder effects linked to migration bottlenecks. Whether or not certain EBV strains are more carcinogenic than others remains in doubt. Early work asking, for example, whether Southeast Asian LMP1 variants had greater transforming potential in in vitro assays than Caucasian variants was inconclusive. More recently interest has been reawakened by the rapid advances in sequencing technology, allowing whole genome analysis of strain identity. The first complete sequence of an NPC tumor-derived EBV strain was published in 2011,59 and many are now following.60,61 One immediate question, yet to be resolved, is whether EBV strains present in the tumors of Southeast Asian patients simply reflect strains prevalent in the population at large or represent a distinct, NPC-associated, subset. Two significant barriers to progress in the field have been the resistance of primary nasopharyngeal cell cultures to stable EBV infection in vitro, thereby limiting studies of the virus’ effects on the normal epithelial phenotype, and the great difficulty of establishing and passaging EBV-positive NPC cells whether as xenografts in immunocompromised mice or as cell lines in culture. The first stable EBV-positive line that could be grown in monolayer culture, C666-1, dates from 199962 and for many years remained the workhorse of in vitro studies. Interestingly both this line and a number of new lines now emerging all derive from the few tumors that were first successfully passaged as xenografts.63,64 No genomically authenticated EBV-positive NPC line has yet been grown direct from fresh tumor tissue despite many attempts, although interestingly there are occasional examples where an authenticated line has grown out but has lost the resident EBV genome.64 The fact that such virus genome loss is never tolerated in vivo emphasizes the continuing contribution EBV must make to the malignant phenotype. Viral markers as screening tools of NPC risk/diagnosis: Ever since its involvement in the serendipitous discovery of the EBV/NPC connection, serology has played an important role in the development of the NPC field from a public health perspective. A key step was made by the Henles who, alerted by a chance observation that total IgA levels were raised in NPC patients,65 showed that the elevation reflected a large virus-specific IgA response66; this was directed toward VCA and often also to the virus-coded early antigen (EA), both actually mixtures of viral proteins expressed in lytically infected cells, as well as to a component of the EBNA complex, later mapped to EBNA1.67 Because such IgA responses were rare in healthy individuals, they were a much better diagnostic marker of incipient NPC than IgG responses, and indeed correlated with tumor burden both at presentation and in response to treatment. The implication was, and is, that the tumor itself is the source of antigens driving this response, yet clear evidence of full lytic replication within the tumor (as opposed to small foci of abortive lytic cycle entry) is still lacking.

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1. NASOPHARYNGEAL CARCINOMA: A HISTORY

These serologic findings have inspired a series of heroic studies, beginning as early as 1982,68 using IgA anti-VCA in combination with IgA anti-EA or similar serologic markers to screen high risk populations, usually 40e60-yearold males in Southern China or Taiwan, in order to identify the small subset of individuals with NPC-like serologic profiles.69,70 Combining data from all surveys with cohort sizes ranging from 1000 to 50,000, a median of some 3% of individuals within that serologically identified subset were found to be carrying an occult NPC, with very few if any cases appearing in the negative population on follow-up; furthermore, the occult tumors thus identified were typically early stage and eminently treatable.71 A more recent and equally interesting approach to screening stemmed from a 1998 paper reporting the presence of EBV DNA in the plasma of 13/42 NPC patients.72 Taking this as a starting point, Dennis Lo and colleagues in Hong Kong refined the virus-specific PCR assay to show that >95% NPC patients were detectably plasma EBV DNA-positive on presentation.73 Furthermore, plasma EBV DNA levels fell rapidly posttreatment, remaining positive in patients with persistent lesions but falling below detection in patients who remained tumor-free and only reappearing as a harbinger of recurrence.74,75 Such plasma-based assays clearly have a role to play in monitoring responses to therapy, but more recent work has investigated their potential in public health screening for NPC. A landmark feasibility study, published in 2017,76 reported the screening of >20,000 Hong Kong males (40e62 years of age), of whom around 300 (1.5%) were consistently plasma EBV DNA-positive on repeat testing. Histologically confirmed NPC was subsequently detected in 34 of these individuals, a positive predictive value of 11%, with the majority having early stage disease; only one individual in the large plasma EBV DNA-negative majority developed NPC within the next year. This clearly illustrates the potential of liquid biopsy technologies for early cancer diagnosis, with NPC currently in the vanguard as proof of principle.

THE IMPACT OF TUMOR GENOMICS As we have seen, NPC is the classic example of a human tumor with multiple cofactors contributing to malignant change. The attempt to understand how those cofactors work together has a long history and is still on-going. However, the debate now occurs against a much richer picture of the oncogenic process, one that has come through identifying the genetic changes seen in NPC cells themselves. It is the study of those genetic changes that we now describe. Tumor cell genomics makes a relatively late appearance in the 50-year history of NPC research (Fig. 1.3) and indeed its most seminal discoveries have come in the last few years. However the story begins in Hong Kong in the late 1980s when Dolly Huang, originally a member of John Ho’s group, committed herself to the study of the NPC cell genome despite the paucity of tools then at her disposal. A first paper in 1989 described the karyotyping of several established xenografts and primary tumors and reported consistent loss of the short arm of chromosome 3 as a key feature of NPC.77 Over the next 5 years her studies also produced evidence of allelic and homozygous deletions on chromosome 9p21 in the majority of NPCs and showed that CDKN2A/p16, a major tumor suppressor on 9p21.3, was inactivated by homozygous deletion or promoter methylation in the tumor.78 Indeed CDKN2A/p16 inactivation by genetic and/or epigenetic routes proved to be one of the most frequent molecular events in NPC tumorigenesis. Many laboratories subsequently joined the field and, from the year 2000 onward, loss-of-heterozygosity (LOH), comparative genome hybridization (CGH) and CGH-based array methodologies all helped to identify multiple common deletion regions (3p14-21, 11q21-qter, 14q24-32, 16q) and also amplifications (11q13, 12p12-13) that were often seen in the NPC genome.79 Thereafter fine mapping within sites of amplification and functional studies identified CCND1/cyclin D1 (on 11q13.3) and LTBR/lymphotoxin receptor B (on 12p13.3) as target oncogenes in 7.3% and 16% of NPC tumors, respectively.80,81 The fact that cyclin D1 amplification impacted on the same growth regulatory pathway as p16 inactivation, both changes favoring uncontrolled cell cycle progression, gave an encouraging logical consistency to the findings. Likewise LTBR encoded a member of the TNF receptor family whose overexpression was associated with NF-kB pathway activation, highlighting a potential synergy with the effects of EBV LMP1. Further studies of candidate tumor suppressor genes in NPC revealed very few examples of inactivation through mutation. However, a long list of genes were found to be epigenetically inactivated by promoter methylation, either in the defined common deletion regions (e.g., RASSF1A, DLEC1, BLU, PTPRG on 3p14-21; TSLC1 on 11q13; MIPOL1 on 14q13; CMTM3, IRF on 16q22-24) or elsewhere in the genome (e.g., DLC1, CHFR, UCHL1, WIF1).82e84 Indeed more recent genome-wide methylome studies have shown the extensive impact of DNA methylation across the NPC genome,85,86 a finding that chimes with analogous findings in EBV-positive cases of another epithelial tumor, gastric carcinoma.87 This has raised the interesting possibility that hypermethylation of the tumor cell genome is

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actually imposed by EBV, particularly as LMP1 expression has been linked to activation of DNA methyl transferase DNMT3b and subsequent gene silencing.88 The revolution in DNA sequencing technology occurring in the past decade has elevated cancer genomics to another level and its transformative effects are now being felt in the NPC field as in many other areas of cancer research. The first landscape study of the NPC genome was made by Koeffler and colleagues in Singapore using whole-exome sequencing (WES) and target capture sequencing.89 This was followed by genome-wide sequencing studies from Hong Kong published in 2016 and 2017.90,91 Together these reports provide a comprehensive catalogue of genetic alterations in a total of 229 NPC samples, thereby greatly extending our understanding of the somatic changes associated with NPC development. The studies have revealed somatic mutations of multiple components in the chromatin modification process (e.g., KMT2D/MLL2, KMT2C/MLL3) and PI3K/MAPK pathways (e.g., PIK3CA, PTEN, NRAS, KRAS) in >25% of the cases. Furthermore, in contrast to the earlier reports of only rare TP53 mutations in NPC, the WES and target capture sequencing studies revealed mutations in 8.5%e10% of NPCs, with greater representation in more advanced disease and in recurrent tumors. Importantly, both of the Hong Kong studies highlighted the high incidence of somatic alterations in a range of genes linked to the inflammatory NF-kB pathways.90,91 Using microdissection to enrich tumor cells, somatic alterations of multiple negative regulators of NF-kB activation, including CYLD, TRAF3, NFKBIA, and NLRC5 were detected in up to 40% of the cases. Most significantly, those mutations occurred in the LMP1-negative subset of tumors and not in tumors with easily detectable levels of LMP1,91 suggesting that activation of NF-kB signaling, a characteristic of all NPCs, may be the result either of cellular genomic change or LMP1 expression from the resident virus genome. Another important finding documented that around 30% of NPCs had lesions in genes involved in the HLA class I pathway of antigen presentation. Furthermore an association between cases with HLA pathway mutations and poor clinical outcome implied that tumor outgrowth benefited from an escape from local CD8þ T cell surveillance.91 While these WES studies have unveiled somatic mutations in the coding regions of key genes, mutations affecting gene regulatory regions (e.g., promoters, enhancers) and structural variations (gene rearrangements, inversions) in NPC genome have not yet been comprehensively explored. In that context, the discovery of recurrent oncogenic fusion genes such as UBR5-ZNF423 and TACC3-FGFR3 suggests an important role for structural variants in NPC tumorigenesis.92,93 Zhao, Kieff, and colleagues in Boston used acetylated histone H3K27ac chromatin immune precipitation followed by deep sequencing (ChIP-seq) to identify novel enhancers and superenhancers in the NPC genome.94 This new epigenetic landscape, and integrative informatics on whole genome sequencing and ChiPseq data, should help to understand the contribution that somatic changes in noncoding regions make to NPC tumorigenesis.

TOWARD AN UNDERSTANDING OF NASOPHARYNGEAL CARCINOMA PATHOGENESIS A comprehensive understanding of NPC pathogenesis has been a goal shared by many laboratories in the field. The challenge was always to show how three cofactors, environment/lifestyle, genetic susceptibility, and EBV infection, might act together to promote malignancy. One of the key questions in that regard was timing. The epidemiological evidence, at least that coming from populations of Cantonese descent, implied the importance of exposure to an environmental carcinogen early in life, with nitrosamines within traditionally preserved salted fish one favored candidate. However, EBV infection also typically occurred during infancy in those populations and so various orders of events were possible. To skeptics of EBV’s involvement, it was even possible that the virus infected NPC cells opportunistically after the malignancy was already established and outgrowth had begun. Such concerns were addressed head-on by the work of Nancy Raab-Traub in Chapel Hill. Using the number of terminal repeats in the circularized EBV episome as a marker of cellular clonality, she showed that every cell in the tumor carried the same episomal size marker. In other words, NPC was a clonal malignancy derived from a single progenitor cell that was already latently infected with EBV.95 Determining when that infection had occurred was especially challenging because preneoplastic lesions of the nasopharynx were extremely rare. Indeed Raab-Traub and colleagues in Malaysia screened >5000 nasopharyngeal biopsies and identified only 11 examples of dysplasia or carcinoma in situ in the absence of invasive NPC. As reported in their 1995 paper,96 all 11 lesions showed evidence of EBV infection and, of those that could be fully evaluated, all had an NPC-like viral latency by transcriptional profiling, all expressed LMP1 by monoclonal antibody staining, and all but one were monoclonal by EBV terminal repeat analysis. This clearly showed that the virus was already present in premalignant lesions, although the fact that five of eight prospectively recorded cases

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went on to develop the tumor in the following year raised the possibility that EBV infection had occurred relatively late during evolution of the malignant clone. These studies were followed by equally important work from Dolly Huang’s lab in Hong Kong, in which normal nasopharyngeal biopsies plus eight rare examples of dysplasia were microdissected and the tissue screened for genetic change by LOH analysis. Two papers published in 2000 and 2002 showed that, as in NPC itself, most of the dysplastic lesions already had LOH at the key chromosomal 3p and 9p loci.97,98 These changes appeared to have preceded EBV infection since the four early stage dysplasias were still EBV-negative while the four late stage lesions were EBV-positive. Such an order of events was further supported by the unexpected finding that, while all histologically normal nasopharyngeal biopsies were negative for EBV markers, >50% of those from Cantonese adults (median age 40 years) had 3p and/or 9p LOH; interestingly the proportion was significantly lower in adult samples from Northern Chinese and was zero in fetal controls.97,98 One possibility, therefore, was that loss/inactivation of key tumor suppressor genes in these chromosomal loci (for example p16 on chr 9p) predisposed the cells either to EBV infection per se or to the stability of such infection. This was indeed demonstrated a decade later by the work of George Tsao and colleagues in Hong Kong. Using immortalized epithelial cell lines of normal nasopharyngeal origin, they found that experimental infection with EBV induced cell senescence and growth arrest; however, knocking down p16 or overexpressing cyclin D1 in the target cells averted this effect and allowed the continued outgrowth of EBV-positive cells with a typical NPC-like pattern of latency.99 Collectively, therefore, these studies have helped to recapitulate some parts of the NPC development pathway, but there is still much to learn. How much can we ascribe the LOH changes seen in the histologically normal nasopharynx of Cantonese people to environmental carcinogens and how much to some inherited predisposition, perhaps linked to one or more of the genetic loci linked to heightened NPC risk? Most of the risk loci reported to date have relatively mild effects, and their identities do not give immediate clues as to their biological significance. The one clear exception, however, is the HLA loci; these have by far the largest effect and their involvement implies a role for immune surveillance restraining tumor development. This is further supported by the more recent finding that up to 30% of tumors have acquired mutations in HLA class I pathway-related genes, in line with earlier reports of reductions or loss of HLA I expression on tumor cells. One possibility is that those HLA alleles that reduce NPC risk, of which HLA-A*1101 is the best example, present antigenic peptides derived from one or more of the EBV antigens expressed in NPC cells; indeed, this is true of A*1101, which in Chinese individuals elicits quite strong CD8þ T cell responses to an LMP2-derived epitope.100 However, it is more difficult to explain the existence of high risk HLA alleles or haplotypes, such as A*0207/Bw46, simply as being poor mediators of relevant CD8þT cell responses when the other alleles in the patient’s HLA type could presumably compensate. It may be that high risk HLA I alleles have an unusually strong influence on innate immune responses, for example, through HLA I-mediated inhibition of NK target cell recognition, and that these innate responses also play a role in tumor surveillance.101 Fig. 1.4 presents a working model of NPC pathogenesis from normal nasopharyngeal epithelium to frank malignancy. Epithelial cells at mucosal surfaces are naturally subject to continual cycles of renewal. We envisage that repeated exposure of cells within the Fossa of Rosenmuller to environmental carcinogens, an insult possibly compounded by chronic inflammation of the nasopharyngeal mucosa,102 greatly increases the risk of genetic alterations occurring. An accumulation of driver events, including inactivation of CDKN2A/p16 and loss of tumor suppressors on chromosome 3p, then promotes the transition from histologically normal epithelium to lowgrade dysplastic lesions, at which point these genetically aberrant epithelial cells are able to retain a chance EBV infection. Note that the incoming virus is likely to have been derived from the B cell reservoir through locally infiltrating B cells reactivating from latency into lytic cycle, epithelial cells being particularly susceptible to infection from the lytically infected B cell surface.103,104 EBV establishes a Latency II-type infection in these dysplastic cells, with expression of EBNA1, LMP1, LMP2A, EBERs, and the BARTs altering multiple cellular signaling pathways to favor cell proliferation, cell survival, and resistance to terminal differentiation. Many of these initial changes would appear to reflect effects induced or magnified by LMP1 in particular. EBV infection also facilitates global hypermethylation of the cell genome, potentially inactivating various tumor-suppressor genes and increasing heterogeneity within the clonally expanding premalignant population. At the same time there will be evolutionary pressure to limit the expression of LMP1, both because of its inherent cytotoxicity when expressed at high levels105 and because of its upregulation of antigen-processing function and thereby immune visibility.106 The frequent loss of LMP1 during NPC progression may be related to acquisition of compensatory genetic alterations by the evolving tumor. That would explain why inactivating mutations in negative regulators in NF-kB pathways are found in LMP1-negative but not LMP1-positive tumors.91 In addition, mutations in HLA class I pathway genes, and multiple components in PI3K/MARK and chromatin remodeling pathways, are selected during tumor evolution. Tumor progression is a continuing process. Thus, in advanced stages, somatic mutations of

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FIGURE 1.4 A working model of nasopharyngeal carcinoma pathogenesis: On a background of underlying genetic susceptibility, exposure to environmental carcinogens induces chromosomal changes in nasopharyngeal epithelium. Precancerous lesions emerge that can sustain latent EpsteineBarr virus (EBV) infection, leading to clonal outgrowth of EBV-positive cells. Thereafter a series of genetic and epigenetic changes occur, leading to tumor formation, progression, and, where treatment fails, recurrence.

TP53, RAS, and other genes may drive subclones of NPC cells to form recurrent and metastatic tumors, thereby acquiring resistance from conventional cancer treatment and allowing regrowth at the local tumor site, within lymph nodes, or in distant organs.

DEVELOPMENT OF NASOPHARYNGEAL CARCINOMA THERAPIES Treatments for NPC have all faced the challenge of dealing with a tumor arising at an anatomically difficult site, the nasopharynx, and often going unrecognized until the appearance of lymph node metastasis. Early attempts at surgery were ineffective and the first steps toward today’s use of radiotherapy (RT) as the bedrock of treatment schedules were initiated almost 100 years ago with New’s report in the Journal of the American Medical Association107 describing the use of radium particles as a local radiation source. This heralded several decades over which the development of increasingly sophisticated linear accelerators ushered in the age of RT as we know it today. In 1963 two linear accelerators arrived at the Queen Elizabeth Hospital in Hong Kong and, under the leadership of John Ho, this became the leading center in Asia, arguably in the world, for the radiation treatment of NPC with accurate staging of the disease a key element in determining radiation schedules.2 Early stage tumors are indeed eminently treatable by RT, though it is still not entirely clear how much this reflects radiosensitivity of the malignant epithelium per se and how much the malignant cell depends upon its radiosensitive lymphoid infiltrate.

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Progress has continued to improve the effectiveness of treatment through a combination of improved beam delivery/scheduling and more accurate staging.108 Thus conventional two-dimensional RT has been superseded in recent years by intensity-modulated RT schedules, where sculpting the high-dose zone allows complete coverage of tumor targets while sparing critical normal structures. Such developments go hand in hand with the extension of staging beyond simple anatomic criteria to include additional factors such as primary tumor volume combined with molecular markers such as plasma EBV DNA levels as a surrogate marker of total tumor mass. However, there is a continuing need for further therapeutic options, given that almost half of all NPC patients present with the disease at an advanced stage, of whom one third will die within 5 years of diagnosis. Encouragingly, a seminal study published by Al-Sarraf and colleagues in 1998109 showed significantly improved 5-year survival for stage III and IV patients given concurrent cisplatin-based chemo/radiotherapy (CRT) compared to RT alone. Several subsequent studies have confirmed the result, making CRT the current standard of choice.108 At the same time, our increasing understanding of NPC pathogenesis has opened up the prospect of entirely novel therapies targeting specific features of the tumor phenotype. A number of different approaches are being explored, typically in small phase II trials. To date, drugs targeting either candidate signaling pathways in NPC cells or the process of angiogenesis have showed little evidence of effectiveness against locoregionally advanced disease.108 To some extent this lack of progress reflects the paucity of laboratory models upon which current candidate drugs can be screened and selected for trial, highlighting the need to expand the number of genomically characterized NPC cell lines and in vivo models available for preclinical studies. A variety of other therapeutic approaches seek to take advantage of NPC’s ubiquitous association with EBV. One such approach is to target the highly methylated viral genome itself, using demethylating agents or other epigenome-targeting drugs in an attempt to reactivate the latent EBV episome into lytic cycle, thereby rendering the cells sensitive to inhibitors of the virus-coded thymidine kinase or to lytic antigen-specific immune recognition.110 Another approach, yet to reach the clinic, aims to target EBNA1, the only viral protein known to be expressed in 100% of tumor cells, using drugs designed to block its DNA binding activity and thereby inhibit its key function, maintenance of the EBV episome and latent infection.111 Perhaps the approaches attracting most current interest are those broadly categorized as immune therapies. The earliest study of this kind dates back to 2001 when four NPC patients were infused with autologous EBV-specific memory T cells that had first been reactivated and expanded in vitro by stimulating with the autologous LCL.112 Subsequent work suggests that this approach, when used in combination with chemotherapy, could be beneficial113 even though reactivities to the limited array of EBV latent antigens expressed in the tumor are only a minor component of LCL-stimulated T cell preparations. To improve specificity, subsequent studies have focused on adoptively transferring T cell preparations enriched for EBNA1 and LMP reactivities and have produced some examples of clinical effect.114 A different route to the same endpoint, vaccinating patients with a recombinant vaccinia virus encoding an EBNA1/LMP2 fusion protein, likewise raised levels of patient immunity to these antigens and gave hints of a clinical response.115 The assumption underlying such work has been that NPC cells remain sensitive to EBNA1/LMPspecific recognition, particularly by CD8þ T cells, and so the variable clinical responses seen in such trials need to be reviewed in light of the finding that a minority of NPCs have genomic changes predicted to impair HLA I-restricted antigen presentation. Most recently, encouraged by results with certain other tumor types, attention has turned to antibody blockade of a candidate immune evasion protein, programmed death ligand-1 PD-L1, which is expressed by most if not all NPCs. Two studies published in 2017/2018 reported that anti-PD-L1 therapy induced a clinical response in 20%e25% of patients,116,117 somewhat lower than seen in some other contexts but still significant. Interestingly in one of these studies the clinical responses appeared to be independent of the tumor’s HLA I status,117 suggesting that such responses may reflect resensitization of the tumor to immune surveillance that is independent of CD8þ T cells.

NASOPHARYNGEAL CARCINOMA: A FUTURE PERSPECTIVE The enduring importance of NPC within the human cancer landscape lies first in its global burden on human health and second in its status as a paradigm of multifactorial cancer development. For both reasons it is a tumor that demands the attention of research groupings that combine multiple disciplines, epidemiology, genetics, medicinal chemistry, virology, immunology, and oncology. Lessons from the past tell us that isolated research laboratories with narrow interests struggle to make progress in this field. Future work toward the two most important goals, improving NPC treatment and ultimately preventing tumor development, will require a multidisciplinary, internationalist approach. To highlight one very simple point, almost all work on NPC has focused on the disease as seen in

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people of Southern Chinese descent and very little on that seen in North African, Inuit, or other ethnic groups. Yet there will be much to learn here from comparative studies that include determinants of genetic susceptibility, the identity of tumor-associated EBV strains, and tumor cell genomics. With respect to global disease burden, NPC is one of two EBV-associated tumors with the highest incidence. Thus there are over 75,000 new NPC cases per year worldwide, the majority involving individuals aged 40e60 years, which is relatively early for cancer development. Furthermore many of these cases present with already established metastatic disease and have a correspondingly poor prognosis. Note that the other main contributor to EBVassociated tumor burden is again a tumor of epithelial origin, namely the distinct subset (variously estimated at 8%e10%) of gastric carcinomas that are EBV genome-positive and occur in all populations worldwide.44 Indeed one of the key objectives for future work in this field should be a much closer integration between groups working on these two different tumors. There will be important lessons to learn both from their similaritiesdfor example, their highly methylated cellular genomes and Latency II patterns of viral infectiondand from their differences, particularly their distinct epidemiologies and patterns of genomic change. There are also important parallels between NPC and some of the EBV-associated lymphoid tumors, for example T/NK cell lymphoma, which typically presents as a pseudoinflammatory lesion in the nasal cavity and Hodgkin lymphoma where, as in NPC, the tumor cells sit within a substantial nonmalignant cell infiltrate.44 How much of that infiltrate represents a host immune response attempting to reject the tumor and how much is induced by the tumor in support of its growth remains a key unresolved question. In terms of new therapies, the combined power of structural biology and medicinal chemistry has huge potential for drug design but the current paucity of preclinical models for drug testing is a serious obstacle to progress. Renewed efforts need to be made toward establishing a wider bank of genomically characterized tumor-derived cell lines and/or xenografts, trying new approaches that build on the pioneering work of Hong Kong and other centers in Southeast Asia. For example, can recent developments in organoid culture provide a way to overcome NPC’s seeming resistance to in vitro passage, and can reconstitution of immunocompromised mice with a hemopoietic system via stem cells from the patient provide a lymphocytic microenvironment conducive to tumor passage in vivo? Tumor prevention may appear a distant goal but, for a virus-associated human malignancy such as NPC, it is in principle achievable. The remarkable success of the human papilloma virus (HPV) vaccine in preventing infection with oncogenic HPV strains is predicted to radically reduce the worldwide incidence of cervical carcinoma over coming decades. Such an achievement shows what can be done with the right mix of science and political will. A prophylactic vaccine capable of preventing EBV infection holds out the prospect of eliminating NPC and all other EBV-associated malignancies. Currently there is a welcome resurgence of interest in this possibility,118 bolstered by recent advances in vaccine design and the identification of vaccine antigens capable of preventing B cell and epithelial cell infection.119,120 The community of NPC researchers, both basic and clinical, must do everything they can to garner support and make this possibility a reality.

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