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VOLUME NINETY THREE
ADVANCES IN
GENETICS
ADVANCES IN GENETICS, VOLUME 93
Serial Editors
Theodore Friedmann
Department of Pediatrics, University of California at San Diego,
School of Medicine, CA, USA
Jay C. Dunlap
Department of Genetics, The Geisel School of Medicine at Dartmouth,
Hanover, NH, USA
Stephen F. Goodwin
Department of Physiology, Anatomy and Genetics,
University of Oxford, Oxford, UK
VOLUME NINETY THREE
ADVANCES IN
GENETICS
Edited by
THEODORE FRIEDMANN
Department of Pediatrics, University of California
at San Diego, School of Medicine, CA, USA
JAY C. DUNLAP
Department of Genetics, The Geisel School
of Medicine at Dartmouth, Hanover, NH, USA
STEPHEN F. GOODWIN
Department of Physiology, Anatomy and Genetics,
University of Oxford, Oxford, UK
AMSTERDAM • BOSTON • HEIDELBERG • LONDON

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First edition 2016
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Notices
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Practitioners and researchers must always rely on their own experience and knowledge in
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ISBN: 978-0-12-804801-6
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CONTRIBUTORS
E.E. Bezsonov
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, MD, United States
H.K. Edskes
T.R. Fenton
UCL Cancer Institute, University College London, London, United Kingdom
A. Gorkovskiy
I.P. Hall
Division of Respiratory Medicine, University of Nottingham, Nottingham, United
Kingdom
Yi-Juan Hu
Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory
University, Atlanta, GA, United States
A.K. Kheirallah
Kingdom
M. Lechner
Head and Neck Centre, University College London Hospital, London, UK; UCL Cancer
Institute, University College London, London, United Kingdom
S. Miller
Kingdom
I. Sayers
Kingdom
E.E. Stroobant
Yan V. Sun
Department of Epidemiology, Rollins School of Public Health, Atlanta, GA, United States;
Department of Biomedical Informatics, School of Medicine, Atlanta, GA, United States
R.B. Wickner
vii j
CHAPTER ONE
The Genomics, Epigenomics,

and Transcriptomics of
HPV-Associated Oropharyngeal
CancerdUnderstanding the
Basis of a Rapidly Evolving
Disease
M. Lechner*, x, 1 and T.R. Fentonx, 1
*Head and Neck Centre, University College London Hospital, London, UK
x
UCL Cancer Institute, University College London, London, United Kingdom
1
Corresponding authors: E-mail: m.lechner@ucl.ac.uk; t.fenton@ucl.ac.uk
Contents
1. Introduction 2
1.1 The Role of Human Papillomavirus in Head and Neck Cancer 3
1.2 Human Papillomavirus-Driven Carcinogenesis 7
2. The Genome of Human Papillomavirus-Associated Head and Neck Cancer 12
2.1 Mutational Processes Shaping the HPVþ HNSCC Genome 13
2.2 Recurrent Mutations in HPVþ HNSCC and Considerations for Targeted Therapy 17
2.3 Copy Number Alterations and Translocations 19
3. The Epigenome of Human Papillomavirus-Associated Head and Neck Cancer 21
3.1 DNA Methylation of the Host Genome in HNSCC 22
3.2 Histone Covalent Modifications and Nucleosomal Remodeling in HPVþ 26
OPSCC
3.3 Epigenetic Modifications of the Viral Genome and Interactions With the Host 27
Genome and Epigenome
3.4 Causative Factors for Epigenetic Changes in HNSCC 28
3.5 Epigenetic Therapy 28
3.6 microRNAs in Head and Neck Cancer 29
4. The Transcriptome of Human Papillomavirus-Associated Head and Neck Cancer 30
4.1 Gene Expression Subtypes in HNSCC 30
4.2 Direct Comparisons of HPVþ and HPV HNSCC Transcriptomes 31
4.3 The Human Papillomavirus Transcriptome in HNSCC 33
4.4 Beyond the Transcriptome: Proteomic Studies of HPVþ HNSCC 34
5. Biomarker Development for Human Papillomavirus-Associated Oropharyngeal 35
Cancer
Advances in Genetics, Volume 93

© 2016 Elsevier Inc.
j
ISSN 0065-2660
http://dx.doi.org/10.1016/bs.adgen.2015.12.001 All rights reserved. 1
2 M. Lechner and T.R. Fenton
6. Linking the Cancer-Associated ChangesdA Model of Human Papillomavirus- 38

Associated Head and Neck Cancer Development
7. Future Directions 38
Acknowledgments 41
References 41
Abstract
Human papillomavirus (HPV) has been shown to represent a major independent
risk factor for head and neck squamous cell cancer, in particular for oropharyngeal
carcinoma. This type of cancer is rapidly evolving in the Western world, with rising
trends particularly in the young, and represents a distinct epidemiological, clinical,
and molecular entity. It is the aim of this review to give a detailed description of
genomic, epigenomic, transcriptomic, and posttranscriptional changes that underlie
the phenotype of this deadly disease. The review will also link these changes and
examine what is known about the interactions between the host genome and viral
genome, and investigate changes specific for the viral genome. These data are then
integrated into an updated model of HPV-induced head and neck carcinogenesis.
1. INTRODUCTION
Head and neck cancer is the sixth most common cancer worldwide
with an incidence of around 600,000 cases per annum, with rising trends
particularly in the young (Hunter, Parkinson, & Harrison, 2005;
Warnakulasuriya, 2009). Despite recent advances in the treatment and in
the understanding of its biology, the 5-year survival rate of 50% for patients
with head and neck cancer on the whole has remained largely unchanged for
the past three decades with relatively few advances since the 1990s (Conley,
2006). Head and neck squamous cell carcinomas (HNSCCs) account for
over 90% of head and neck cancers and are the focus of this review.
As an umbrella term, HNSCC includes cancers arising in mucosal
epithelia at several locations of the upper aerodigestive tract, eg, in the
oral cavity, pharynx, and larynx. Although these cancers have different
etiologies and prognoses, they share similar risk factors. These major risk
factors include the smoking and chewing of tobacco as well as alcohol
consumption (Hunter et al., 2005). Heavy smokers under the age of 46
have a 20-fold increased risk of oral and pharyngeal cancer, whereas heavy
drinkers are reported to have a 5-fold increased risk (Rodriguez et al., 2004).
A combination of heavy smoking and drinking leads to an almost 50-fold
increased risk of oral and pharyngeal cancer (Rodriguez et al., 2004).
OPSCC Genomics 3
However, a proportion of HNSCC patients (up to 20%) have little or no

tobacco or alcohol exposure, indicating other risk factors for the disease.
1.1 The Role of Human Papillomavirus in Head and Neck

Cancer
Human papillomavirus (HPV) was recognized as the cause of cervical cancer
and small numbers of anogenital cancers in the 1980s, and in 2007 the
World Health Organization’s International Agency for Research on Cancer
recognized HPV as a major independent risk factor also for HNSCC
(D’Souza et al., 2007; Tran, Rose, & O’Brien, 2007). HPV is particularly
associated with oropharyngeal carcinoma (squamous cell cancer of the tonsils
and base of tongue). More than 50% of these tumors test positive for the
HPV16 subtype, and in some countries the numbers have risen to over
90% in certain age groups (D’Souza et al., 2007; Mendenhall & Logan,
2009; Nasman et al., 2009; Tran et al., 2007). HPV-positive (HPVþ)
HNSCC appears distinct from HPV-negative (HPV) HNSCC with
regard to mutational and gene expression profiles as well as to epidemiolog-
ical factors and clinical features, leading to the conclusion that HPVþ
HNSCC represents a distinct molecular, epidemiological, and clinical entity
(Gillespie, Rubinchik, Hoel, & Sutkowski, 2009; Gillison, 2004).
HPVþ HNSCC is a rapidly emerging disease in the developed world
with rising trends particularly among the young. There are currently
10,000 cases in the US alone each year and this number is projected to
rise to 16,000 by 2030, a trend unlikely to be reversed by prophylactic vacci-
nation until at least 2060 (Gillison, Chaturvedi, Anderson, & Fakhry, 2015).
Both HPVþ and HPV HNSCCs occur more frequently in men at a rate
of three to one compared to women (Hunter et al., 2005; Warnakulasuriya,
2009). The increasing rate of squamous cell cancer of the tonsils and of the
tongue base is particularly evident in patients under the age of 45 and this
group has experienced a 2% per year increase in base of tongue cancer
and a 4% per year increase in tonsillar cancer between 1973 and 2004. In
contrast, the rates for cancers at all other head and neck sites during the
same period remained constant or decreased (Sturgis & Cinciripini, 2007).
This trend is also underscored by a further study investigating archived tonsil
cancer specimens: HPV DNA was isolated in 23% of the specimens from the
1970s, 28% of the specimens of the 1980s, 57% from the 1990s, and 68% of
specimens since 2000 (Hammarstedt et al., 2006). A recent study by the same
group suggests that the incidence of HPVþ tonsillar carcinoma has increased
further still from 2003 to 2007 (with 68% 2000e02 to 93% 2006e07) with a
parallel decline of HPV tumors. Taking into account the previously pre-
sented data, the incidence of HPV-positive tonsillar tumors has almost
doubled each decade between 1970 and 2007, suggesting an epidemic of
viral-induced carcinoma (Nasman et al., 2009).
HPV-related HNSCC occurs more frequently in younger patients (age
<50 years) with minimal tobacco and/or alcohol exposure but greater
exposure to oral sex, multiple sexual partners, or marijuana (Gillison et al.,
2008). Compared to HPV HNSCC patients, HPVþ HNSCC patients
are often of higher socioeconomic status with better nutrition, dentition,
and overall health compared to patients suffering from HPV disease.
Racial differences for HPV-related HNSCC have also been demonstrated.
In their study, Settle et al. (2009) found that 35% of Caucasian patients
developed HPVþ tumors compared with only 4% of patients of African
origin.
In summary, the epidemiological trend suggests that HPV-related
oropharyngeal squamous cell cancer is becoming more widespread at a
time when the overall incidence of HNSCC is falling and this subtype typi-
cally occurs in younger patients (age <50 years).
Moreover, this has major clinical implications. HPVþ HNSCC responds
better to chemotherapy and radiotherapy (82% vs 55% response rate for
HPV cases) and has a better disease-free and overall survival (95% vs
62% at 2 years) (Fakhry et al., 2008). Individuals with HPVþ HNSCC
have a lower rate of second primary tumors, as well as a decreased cumula-
tive incidence of relapse (Hafkamp et al., 2008; Licitra et al., 2006). Thus,
knowledge of a patient’s HPV status offers the possibility to stratify such
patients for treatment and to elucidate the mechanisms underlying the
virus-associated advantage in drug response and survival in HNSCC. A
summary of the distinct molecular, epidemiological, and clinical features is
provided in Table 1.
The causes responsible for the different clinical behaviors of HPVþ and
HPV tumors remain poorly understood. Aside from the differences in age
and overall health of patients noted above, molecular differences between
HPVþ and HPV HNSCCs, including mutational profiles and overall
mutation burden, intratumoral heterogeneity, epigenomic and transcrip-
tional profiles, and the infiltration of tumor-reactive lymphocytes may all
play a role in the differing clinical behavior of these tumors. This review
will focus on the considerable progress that has been made in the identifica-
tion of these molecular differences in the past few years and on the potential
for translation of this knowledge into improved outcomes for patients.
OPSCC Genomics
Table 1 Distinct molecular, epidemiological, and clinical features of HPVþ HNSCC, compared with HPV HNSCC
HPVþ HNSCC HPV HNSCC
Molecular factors TP53 wild type present in most cases TP53 mutational loss (disruptive) common
(if TP53 mutations are present, they are (Westra et al., 2008)
usually not disruptive (Westra et al., 2008)) CDKN2A(p16) underexpression (Jung
TP53 loss of function (rendered by the et al., 2010) or mutational loss (Jung et al.,
viral oncoprotein E6) (Mendenhall & 2010; Stransky et al., 2011)
Logan, 2009). No HPV DNA/RNA
RB1 loss of function (rendered by the viral
oncoprotein E7) (Doorbar, 2006)
CDKN2A(p16) overexpression (Jung
et al., 2010)
HPV DNA (type 16 in up to 85% of cases)
(D’Souza et al., 2007; Herrero et al., 2003;
Kreimer et al., 2005)
Possible epidemiological Often nonsmokers (D’Souza et al., 2007; Heavy smokers (D’Souza et al., 2007;
factors Gillison et al., 2008) Gillison et al., 2008)
Mild/moderate alcohol intake (D’Souza Heavy alcohol intake (D’Souza et al.,
et al., 2007; Gillison et al., 2008) 2007; Gillison et al., 2008)
High marijuana exposure (Gillison et al., Low marijuana exposure (Gillison et al.,
2008) 2008)
Intact dentition (Gillison et al., 2008) Poor dentition (Gillison et al., 2008)
High oral sex exposure (D’Souza et al., No Association with sexual behaviors
2007; Gillison et al., 2008) (D’Souza et al., 2007; Gillison et al., 2008)
(Continued)
5
Table 1 Distinct molecular, epidemiological, and clinical features of HPVþ HNSCC, compared with HPV HNSCCdcont'd
6
HPVþ HNSCC HPV HNSCC
Younger age (<45 years) (Chaturvedi, Older age (>50 years) (Chaturvedi et al.,
Engels, Anderson, & Gillison, 2008; 2008; Chaturvedi et al., 2011)
Chaturvedi et al., 2011) Decreasing incidence of the disease (Marur
Increasing incidence of the disease (Marur, et al., 2010; Nasman et al., 2009)
D’Souza, Westra, & Forastiere, 2010;
Nasman et al., 2009)
Histopathological Frequently basaloid, nonkeratinizing, Frequently keratinizing SCC (Mendelsohn
appearances poorly differentiated SCC (Mendelsohn et al., 2010)
et al., 2010)
Clinical factors Predominantly oropharynx (tonsil and All head and neck sites (D’Souza et al.,
tongue base) (D’Souza et al., 2007; 2007; Mendenhall & Logan, 2009; Tran
Mendenhall & Logan, 2009; Tran et al., et al., 2007)
2007) Worse survival (Ang et al., 2010; Fakhry
Better survival (Ang et al., 2010; Fakhry et al., 2008)
et al., 2008) Radiation response unpredictable (Fakhry
More radiosensitive (Fakhry et al., 2008) et al., 2008)
Higher rates of patients presenting with
nodal metastases and SCC of unknown
primary (Jensen et al., 2014; Mendelsohn
M. Lechner and T.R. Fenton

et al., 2010)
HPVþ HNSCC, human papillomavirus-positive head and neck squamous cell carcinoma.
Adapted from Gillespie, M.B., Rubinchik, S., Hoel, B., & Sutkowski, N. (2009). Human papillomavirus and oropharyngeal cancer: what you need to know in 2009.
Current Treatment Options in Oncology, 10(5e6), 296e307. http://dx.doi.org/10.1007/s11864-009-0113-5.
OPSCC Genomics 7
1.2 Human Papillomavirus-Driven Carcinogenesis

HPVþ HNSCC is clearly characterized by molecular pathways distinct from
those involved in HPV HNSCC (Gillison, 2004). In particular, the central
role of the high-risk HPV oncoproteins (E6 and E7) has been studied
extensively in relation to cervical cancer, and much of this knowledge is
directly applicable to the oropharynx (Egawa, Egawa, Griffin, & Doorbar,
2015). Here we briefly summarize the HPV life cycle, what is known
regarding HPV-driven carcinogenesis and how this applies to HPVþ
HNSCC. For more information the reader is referred to detailed reviews
on this subject (Doorbar et al., 2012; Groves & Coleman, 2015).
HPVs are ubiquitous, nonenveloped, circular, double-stranded DNA
viruses. Over 200 HPV types have been isolated (http://pave.niaid.nih.
gov) and it is believed that HPV represents the most common cause of
cancers induced by an infectious agent (zur Hausen, 2009; Van Doorslaer
et al., 2013). Of over 40 known types of HPV that are typically transmitted
by sexual contact, the World Health Organization currently lists 12 as group
1 carcinogens due to their clear causative role in cancer and recent data
support the inclusion of a further 8 types in this category (Halec et al.,
2014). Of these “high-risk” types, HPV16 and 18 together account for
approximately 70% of cervical cancers worldwide, with other high-risk
HPVs accounting for most of the remaining 30% of cases. For reasons we
do not yet understand, HPV16 is very dominant in HPV-associated head
and neck cancers, accounting for over 85% of cases (Kreimer, Clifford,
Boyle, & Franceschi, 2005).
The approx 8000 bp HPV genome contains eight genes (Fig. 1A) whose
expression is tightly linked to differentiation, allowing HPV to complete its
productive life cycle in stratified epithelia (Fig. 1B). Depending upon the
anatomical subsite at which the infection occurs, virus access to the target
cells in the basal epithelium may occur via several routes. In some cases
this cell layer is directly exposed to the lumen (eg, at the cervical or anal
transformation zones). It is also possible that the virus gains entry through
microabrasions in the stratified epithelium. In the tonsillar crypts, the retic-
ulated nature of the epithelium may allow direct access of the virus to the
basal layer (Egawa et al., 2015, see Fig. 1B). Following infection of cells
in the basal layer of stratified epithelia, the viral genome is maintained as a
circular extrachromosomal element (nuclear episome) at typically around
50e100 copies per cell. The E6 and E7 early genes are expressed in these
cells, in the case of high-risk types triggering cell cycle entry and replication
Figure 1 HPV16 genome organization and life cycle. (A) The prototypical HPV16
genome showing the viral oncogenes (E6 and E7, also known as accessory genes, since
they are not found in all papillomaviruses) in red (gray in print versions), the genes
required for viral genome replication and amplification (E1, E2, E4, and E5) in yellow
(light gray in print versions) and the capsid genes (L1 and L2) in green (dark gray in
print versions). The long control region is shown upstream of the p97 promoter,
from which the bicistronic E6/E7 transcript is expressed. E1eE5 are expressed from
the p670 promoter, which also drives expression of L1 and L2 following activation of
alternative splicing in the upper layers of the epithelium. Lollypops represent sites of
OPSCC Genomics 9
of both cellular and viral DNA by the host DNA polymerase machinery. As
cells move up from the basal layer and begin to differentiate, E6/E7 expres-
sion decreases and the remaining early genes (E1, E2, E4, and E5) are tran-
scribed from a second promoter. These genes drive viral genome
amplification to greater than 1000 copies per cell. Finally, as cells reach ter-
minal differentiation in the uppermost layer the capsid proteins (L1 and L2)
are expressed and progeny virions are assembled. These virions are then
released as the host cells die (Fig. 1B, also see (Doorbar et al., 2012; Egawa
et al., 2015) and references therein for further detail).
Doorbar and colleagues have proposed that problems occur when high-
risk HPVs infect cells with two key characteristics. Firstly, stem, or stemlike
cells (such as cervical reserve cells), in which the virus can persist with min-
imal viral gene expression for many years without the host cell entering into
epithelial differentiationda state increasingly recognized as HPV latency
(Doorbar, 2013). Secondly, cells in which the correct regulation of viral
gene expression does not occur, and therefore a productive viral life cycle
is not supported. In the oropharynx, the reticulated epithelium of the
tonsillar crypts may represent such a subsite, with the additional consider-
ation that virally infected stem cells may escape surveillance in this in this
=---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
CpG methylation that have been detected in tumor cells (see Section 3.3). (B) Schematic
representation of a productive HPV16 infection in the tonsillar crypt. (1) Viral entry to
the basal layer may be facilitated by the reticulated nature of the stratified epithelium
at this subsite. (2) Upon entry into the nuclei of basal progenitor cells, E6 and E7 are
expressed, causing cell cycle entry and cell division in the basal layer. (3) As cells
move up through the epithelium and begin to differentiate, E1, E2, E4, and E5 are
expressed from the p670 promoter and viral genome amplification occurs. (4) Alterna-
tive splicing in the terminally differentiated cells of the upper squamous epithelium
produces the L1 and L2 capsid proteins, allowing viral packaging and shedding of virus
as the differentiated keratinocytes die and slough away from the epithelium. (C) Rep-
resentation of an invasive SCC in the tonsillar crypt epithelium. Regulation of viral gene
expression has been lost, resulting in constitutive, high-level E6/E7 production, inhibi-
tion of differentiation and extensive proliferation of cells throughout the epithelium.
Additional alterations to the host genome and epigenome cooperate to generate an
invasive, metastatic phenotype. The tumor has invaded through the basement mem-
brane into the lymphoid tissue below and tumor infiltrating lymphocytes are evident
in the tumor mass. Figure adapted from Doorbar, J., Quint, W., Banks, L., Bravo, I.G.,
Stoler, M., Broker, T.R., & Stanley, M. A. (2012). The biology and life-cycle of human pap-
illomaviruses. Vaccine, 30(Suppl. 5), F55eF70. http://dx.doi.org/10.1016/j.vaccine.2012.06.
083. and Egawa, N., Egawa, K., Griffin, H., & Doorbar, J. (2015). Human papillomaviruses;
epithelial tropisms, and the development of neoplasia. Viruses, 7(7), 2802. Retrieved from:
http://www.mdpi.com/1999-4915/7/7/2802 with kind permission from Dr. John Doorbar.
immune-privileged lymphoid tissue (Egawa et al., 2015). Development of

invasive SCC in the tonsillar epithelium following deregulation of E6/E7
expression and additional somatic alterations to the host genome and
epigenome (see later) is shown in Fig. 1C.
The oncogenic activities of the high-risk papillomaviruses are largely
attributable to two viral early genes, E6 and E7. These “oncoproteins”
degrade and destabilize two major tumor suppressor proteins, tumor protein
p53 (p53) and retinoblastoma 1 (pRB). E6 brings p53 into complex with an
E3 ubiquitin ligase (UBE3A, also referred to as E6-associated protein
(E6AP)), resulting in its ubiquitination and targeting for degradation by
the proteasome (Huibregtse, Scheffner, & Howley, 1991; Scheffner,
Huibregtse, Vierstra, & Howley, 1993; Scheffner, Werness, Huibregtse,
Levine, & Howley, 1990). Loss of p53 results in deregulation of both the
G1/S and G2/M cell cycle checkpoints during cell damage and other
cellular stress, which leads to genomic instability (Kessis et al., 1993). The
HPV E7 oncoprotein binds pRb and induces its degradation by recruitment
of the cullin 2 ubiquitin ligase complex (Dyson, Howley, Munger, &
Harlow, 1989; Huh et al., 2007; Munger et al., 1989). Both binding of
pRb by E7, and its subsequent proteasomal degradation liberate E2F family
transcription factors, resulting in the activation of S-phase genes and cell
proliferation (Slebos et al., 1994).
In addition to their well-studied effects on p53 and pRb, E6 and E7
interact with and modulate the activity of numerous other host proteins.
Many of these interactions impinge upon critical regulatory networks and
may allow the infected cell to acquire many of the hallmarks of cancer as
proposed by Hanahan and Weinberg (Fig. 2 (Hanahan & Weinberg,
2011; Leemans, Braakhuis, & Brakenhoff, 2011; Mesri, Feitelson, &
Munger, 2014; Pim & Banks, 2010)). Of particular interest to carcinogenesis
are those proteineprotein interactions that are specific to or enhanced in the
oncoproteins encoded by high-risk HPV types. One such feature is the C-
terminal PDZ (PSD95/Dlg/Zo-1) domain-binding motif that is found only
in high-risk E6 proteins and is required E6-driven tumorigenesis in mice
(Pim & Banks, 2010). PDZ domains are found in a number of proteins
involved in maintaining apical-basal polarity and cellecell junctions, dysre-
gulation of which characterizes the epithelial to mesenchymal transition
(EMT), during which carcinoma cells acquire an invasive, metastatic pheno-
type. E6 targets many other genes in a p53-independent manner, including
the proapoptotic BCL2-antagonist/killer 1 (BAK1), cyclin-dependent kinase
inhibitor 1A (CDKN1A; also referred to as p21 or Cip1), telomerase
OPSCC Genomics 11
Figure 2 Interaction of the HPV16 E6 and E7 oncoproteins with multiple cellular

proteins results in the acquisition of cancer hallmark characteristics in the host cell. In
addition to their well-characterized effects on p53 and pRb, E6 and E7 interact with
and/or regulate multiple other cellular targets, some of which are shown here. A num-
ber of these interactions are critical for E6/E7-dependent transformation in cell and an-
imal models. Here, the potential roles of these interactions on carcinogenesis are
represented by overlaying them onto a number of the hallmarks of cancer described
by Hanahan & Weinberg (2011). For further details, see Section 1.2 and Leemans, C.R.,
Braakhuis, B.J., & Brakenhoff, R.H. (2011). The molecular biology of head and neck cancer.
Nature Reviews Cancer, 11(1), 9e22. 10.1038/nrc2982, Mesri, E.A., Feitelson, M.A., &
Munger, K. (2014). Human viral oncogenesis: a cancer hallmarks analysis. Cell Host &
Microbe, 15(3), 266e282. 10.1016/j.chom.2014.02.011, and Pim, D., & Banks, L. (2010).
Interaction of viral oncoproteins with cellular target molecules: infection with high-risk vs
low-risk human papillomaviruses. APMIS, 118(6e7), 471e493. http://dx.doi.org/10.1111/j.
1600-0463.2010.02618.x.
(hTERT), and the closely related transcriptional coactivators p300 and CREB
binding protein (EP300 and CREBBP) (Leemans et al., 2011; Mesri et al.,
2014; Pim & Banks, 2010). Through their interactions with these and other
targets (for a detailed review see (Pim & Banks, 2010)), E6 and E7 therefore
set the stage for cellular transformation and to a large extent, likely dictate the
additional genetic and epigenetic alterations that are necessary for the devel-
opment of invasive carcinoma. Recent large-scale -omics studies have begun
to reveal some of the most common somatic events seen in HPVþ HNSCC
and these will be discussed in detail in Sections 2 and 3.
Integration of viral genes is frequently observed in HPV-associated cancers
(Kim et al., 2007; Parfenov et al., 2014; Schmitz, Driesch, Jansen, Runne-
baum, & Durst, 2012). The E2 gene, which functions in HPV genome repli-
cation and normally acts as a repressor of E6 and E7 transcription, is often
disrupted upon integration. Complete loss of E2 expression occurs following
the clearance of remaining episomes and results in a significant increase in E6/
E7 expression in cervical keratinocytes undergoing HPV-driven transforma-
tion in culture (Pett et al., 2006). The overlapping E5 gene is also commonly
lost from integrants, and as such is not regarded as being a mediator in late
stage carcinogenesis, although it may play important roles in the initiation
of cellular transformation (see Section 4.3 (Schwarz et al., 1985)). Mapping
of HPV integration sites by whole-genome sequencing (WGS) in HPVþ
HNSCC revealed a bias toward genic regions, frequently associated with dis-
rupted expression and/or rearrangements of host genes, suggesting this may
be a contributing factor in the development of these tumors (Parfenov
et al., 2014). Integration-independent transformation of HPV-infected kera-
tinocytes has been observed in culture, however, and in The Cancer Genome
Atlas (TCGA) WGS study, 10 of 35 HPVþ HNSCCs showed no evidence of
integration, thus integration is not a requirement for HPV-associated tumor
development (Gray et al., 2010). Interestingly, HNSCCs with integrated
HPV and those without integration display distinct gene expression profiles
and DNA methylation patterns, suggesting they may arise via subtly different
mechanisms and may potentially display vulnerabilities to different therapeu-
tic approaches (Parfenov et al., 2014).
2. THE GENOME OF HUMAN PAPILLOMAVIRUS-

ASSOCIATED HEAD AND NECK CANCER
As described above, deregulated expression of E6 and E7 initiates
tumorigenesis by abrogating key tumor suppressor pathways, but full cellular
OPSCC Genomics 13
transformation requires the acquisition of lesions in the host genome, hence

the considerable latency between infection and a diagnosis of invasive carci-
noma in both cervical cancer and HPV-associated HNSCC (reviewed in
(Goon et al., 2009; M€ unger et al., 2004)). As with all tumor types, the
advent of massively parallel next generation sequencing has revolutionized
our ability to probe the genomic aberrations underlying HNSCC. Two
landmark whole-exome sequencing (WES) studies published in 2011 gave
important insight into the genomics of HPV HNSCC but only 11 of a to-
tal 106 tumors sequenced by the two groups were HPVþ (Agrawal et al.,
2011; Stransky et al., 2011). These studies suggested a lower overall muta-
tional burden in HPVþ disease and together with a further whole-exome
study and targeted sequencing of cancer gene panels, implicated mutational
activation of the phosphoinositide 30 -kinase (PI3K) pathway as a key feature
of these tumors (Agrawal et al., 2011; Chung et al., 2015; Lechner,
Frampton, et al., 2013; Lui et al., 2013; Stransky et al., 2011). Our under-
standing of HPVþ HNSCC genomes and the mutational processes that
shape them has been greatly enhanced by the comprehensive analysis of
approximately 300 head and neck squamous carcinomas by
TCGA project, over 40 of which are HPVþ (The Cancer Genome Atlas,
2015). Here we will discuss how our new knowledge of HPVþ HNSCC
genomics has helped to advance our understanding of HPVþ tumor evolu-
tion and therapeutic opportunities.
2.1 Mutational Processes Shaping the HPVþ HNSCC

Genome
In HPV HNSCC there is a clear link between risk factors (heavy tobacco
smoking and alcohol consumption) and somatic mutagenesis: many compo-
nents of tobacco smoke cause the formation of bulky adducts on DNA bases,
particularly guanine, which if not repaired correctly result in characteristic
point mutations at G:C base pairs. Likewise, melanomas display mutations
that are characteristic of the mutagenic process underlying the disease (in
this case, UV-induced formation of pyrimidine dimers) (Helleday, Eshtad,
& Nik-Zainal, 2014). Until recently, it was not clear whether a specific
mechanism acts to drive somatic mutagenesis during the development of
HPVþ HNSCC, where patients often have no tobacco smoking history
for instance. Recent developments in computational methods, however,
particularly the use of nonnegative matrix factorization (NMF) to deconvo-
lute complex data sets into specific patterns, have enabled the detection of
these “mutational signatures” in cancer sequence data, allowing us to
uncover a number of mutagenic processes at play during tumor develop-

ment (Alexandrov, Nik-Zainal, Wedge, Aparicio, et al., 2013; Alexandrov,
Nik-Zainal, Wedge, Campbell, & Stratton, 2013).
Application of NMF to WES data from 298 HNSCCs sequenced by
TCGA reveals three major mutational signatures ((Henderson, Chakravarthy,
Su, Boshoff, & Fenton, 2014), Fig. 3). Not surprisingly given the known risk
factors for HNSCC, a mutational signature that has been linked to tobacco
smoking is present. This signature, which is also seen in lung and liver
cancers is characterized by C:G > A:T transversions, largely independent
of trinucleotide context (Alexandrov, Nik-Zainal, Wedge, Aparicio,
et al., 2013). As described in the previous paragraph, these mutations result
from errors in repair following the addition of bulky adducts to guanine
bases. Also detectable is a signature characterized by C > T transitions at
CpG sites. This signature is seen in many cancers and is thought to result
from the spontaneous deamination of methylated cytosine bases during
agingdalso a risk factor for HNSCC (Alexandrov, Nik-Zainal, Wedge,
Aparicio, et al., 2013). The third signature is characterized by C > T
transitions and C > G transversions, at TpC sites, predominantly in the
TpCpA/T (TCW) trinucleotide context (Fig. 3). The nature of these
mutations and their specific sequence context suggests that they result
from enzymatic cytosine deamination catalyzed by one or more apolipo-
protein-B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)
cytosine deaminases (Alexandrov, Nik-Zainal, Wedge, Aparicio, et al.,
2013; Burns, Lackey, et al., 2013; Nik-Zainal et al., 2012; Roberts et al.,
2012). The 11-gene APOBEC family includes the activation-induced
deaminase (AID), which catalyzes somatic hypermutation and class switch
recombination of immunoglobulin genes in B-cells via the deamination of
cytosines to uracils. Normally, genomic uracil is recognized and removed
by uracil DNA glycosylase and cytosine is restored by base excision repair.
However, this process can be interrupted by DNA replication, during
which translesion DNA polymerases pair dA opposite dU, or if the uracil
has been removed leaving an abasic site, dA or dC are most frequently
incorporated on the daughter strand, resulting in C > T or C > G muta-
tions in the subsequent round of replication (mutation fixation) (Di Noia &
Neuberger, 2007). AID is not generally expressed outside of the B-cell
lineage, however, and does not display a preference for TpC sites. Instead,
it appears that during tumor development, one or more members of the
7-gene APOBEC3 (A3) family can become mistargeted toward genomic
DNA, resulting in the characteristic mutational signature that is particularly
OPSCC Genomics
Figure 3 Mutational signatures in head and neck squamous cell carcinoma (HNSCC) tumors sequenced by Cancer Genome Atlas (TCGA). Ap-
plication of nonnegative matrix factorization to whole-exome sequence data from the TCGA HNSCC analysis reveals three major mutational
signatures that can be attributed to APOBEC activity, age, and tobacco smoking. HPVþ, human papillomavirus-positive. Figure adapted from
15
Henderson, S., Chakravarthy, A., Su, X., Boshoff, C., & Fenton, T.R. (2014). APOBEC-mediated cytosine deamination links PIK3CA helical domain
mutations to human papillomavirus-driven tumor development. Cell Reports, 7(6), 1833e1841. http://dx.doi.org/10.1016/j.celrep.2014.05.012.
prominent in carcinomas of the bladder, cervix, head and neck, breast, and
lung (Alexandrov, Nik-Zainal, Wedge, Aparicio, et al., 2013; Burns,
Lackey, et al., 2013; Burns, Temiz, & Harris, 2013; Roberts et al.,
2013). Several A3 (A3AeD, F, G, H) enzymes function in innate
immunity, creating C > U mutations in single-stranded viral DNA. The
restriction of HIV infectivity by A3G in T-lymphocytes is the best-studied
example of this but HPV DNA is also targeted by A3s and recent data
suggest A3A could function as an HPV restriction factor (Vartanian,
Guetard, Henry, & Wain-Hobson, 2008; Warren et al., 2015). Stratifica-
tion of the TCGA HNSCC data set by HPV status based on detection
of RNA-seq reads corresponding to viral transcripts revealed increased
expression of A3B and strong enrichment for the APOBEC mutational
signature in the HPVþ tumors (Fig. 3 (Henderson et al., 2014)). As
discussed above, the PI3K pathway is subject to recurrent mutational
activation in HNSCC. In particular, PIK3CA, which encodes the catalytic
PI3K isoform, p110a, is mutated and/or amplified in over 50% of HPVþ
HNSCC (The Cancer Genome Atlas, 2015). Strikingly, PIK3CA
mutations in HPVþ HNSCC almost invariably occur at one of two
hotspots in the helical domain, which is responsible for the interaction
with the p85 adapter subunit. These glutamate to lysine mutations
(E542K and E545K) both result from C:G > T:A transitions within
optimal (TpCpA) motifs for cytosine deamination by A3. In HPV
HNSCC, PIK3CA mutation is also frequent but often these mutations
are at a second hotspot (H1047) in the kinase domaindthese are typically
A:T > G:C transitions, unrelated to cytosine deamination (Henderson
et al., 2014). The correlation between A3 signature mutations and helical
versus kinase domain PIK3CA mutations across multiple cancer types and
the occurrence of these mutations coincident with appearance of the A3
signature in lung cancer subclones during tumor evolution both strongly
suggest that PIK3CA helical domain mutations are caused by A3 activity
(de Bruin et al., 2014; Henderson et al., 2014). These findings, confirmed
by TCGA’s own analysis of the data and supported by studies showing that
A3B is induced by E6 in keratinocytes following HPV infection, suggest a
model in which HPV oncogene expression not only creates genomic
instability but also acts in a hitherto unanticipated fashion to drive somatic
mutagenesis of key protooncogenes by triggering APOBEC activity
((Henderson et al., 2014; The Cancer Genome Atlas, 2015; Vieira et al.,
2014; Warren et al., 2015) reviewed in (Henderson & Fenton, 2015)).
OPSCC Genomics 17
2.2 Recurrent Mutations in HPVþ HNSCC and

Considerations for Targeted Therapy
In HNSCC, we are able to study squamous cell carcinomas that arise at
common anatomic sites with distinct viral and nonviral etiologies, affording
a unique opportunity to learn more about the core pathways required for
tumor development and how they become deregulated. Early neoplastic
events in up to 80% of HPV HNSCC include the mutational loss of
cyclin-dependent kinase inhibitor 2A (CDKN2A; which encodes two
tumor suppressor proteins, p16INK4A and p19ARF through the use of
alternative splicing) and TP53, leading to uncontrolled cellular growth
(Califano et al., 1996). When the analysis is restricted to oropharyngeal squa-
mous cell carcinoma (OPSCC), TP53 mutation is an almost universal
feature of HPV disease while in contrast, TP53 mutations are very rarely
seen in HPVþ HNSCC, suggesting that while loss of p53 function is oblig-
atory for OPSCC development, degradation of p53 by E6 removes selective
pressure for its genetic ablation in these tumors (Agrawal et al., 2011;
Lechner, Frampton, et al., 2013; Stransky et al., 2011; The Cancer Genome
Atlas, 2015; Westra et al., 2008). Likewise, genes involved in G1/S check-
point control remain unperturbed in HPVþ HNSCC, while CCND1
amplification or CDKN2A loss/mutation are commonly seen in HPV
HNSCC. The degradation of p53 and abrogation of pRb function are
well-characterized properties of the HPV16 E6 and E7 proteins but some
of the other mutations exclusive to HPV HNSCC suggest an important
role for lesser-studied facets of E6/E7 biology in squamous carcinogenesis.
One potential example of this is the observation that the Wnt pathway regu-
lator FAT1 is mutated at high frequency (32%) in HPV but only rarely
(3%) in HPVþ HNSCC (The Cancer Genome Atlas, 2015). FAT1 encodes
a cadherin-like protein that binds b-catenin, anchoring it at the plasma
membrane, a function that is lost in cancer-derived FAT1 mutants, resulting
in nuclear translocation of b-catenin and activation of downstream gene
expression (Morris et al., 2013). In HPVþ OPSCC cells Wnt signaling is
activated in an E6/E7-dependent manner, potentially through transcrip-
tional repression of the SIAH1 ubiquitin ligase, resulting in b-catenin stabi-
lization (Rampias et al., 2010). An alternative mechanism for upregulation of
Wnt signaling by HPV16, independent of effects on b-catenin stability but
mediated through the E6-E6AP interaction has also been proposed (Lichtig
et al., 2010). As with p53 and the G1/S checkpoint, it may be that HPV
oncogene expression phenocopies and therefore obviates selection pressure
for Wnt pathway mutations during HNSCC development. Wnt signaling

regulates cell fate in close cooperation with the Notch pathway, which pro-
motes differentiation in squamous epithelia and is inactivated by NOTCH1
mutations in both HPVþ and HPV HNSCC (Agrawal et al., 2011; The
Cancer Genome Atlas, 2015). Another pathway that is mutated in HPV
HNSCC but not in HPVþ is oxidative stress response, either directly
through mutational activation of NFE2L2, which encodes the NRF2 tran-
scription factor or through inactivation of either KEAP1 or CUL3, both of
which function in the ubiquitin-mediated degradation of NRF2 (Leinonen,
Kansanen, P€ ol€
onen, Hein€aniemi, & Levonen, 2014; The Cancer Genome
Atlas, 2015). Whether redox signaling differs between HPV and HPVþ
HNSCCs and how HPV might modulate the oxidative stress response is
unclear.
The most frequently mutated gene in HPVþ HNSCC is PIK3-
CAIndeed in some HPVþ tumors PIK3CA or the PIK3R1 gene encoding
the p85a adapter subunit of the PI3K heterodimer are the only known
cancer genes that harbor mutations, suggesting a potentially targetable addic-
tion to this pathway (Lui et al., 2013). As discussed in Section 2.1, PIK3CA
mutations in HPVþ HNSCC almost always occur in the helical domain
(Henderson et al., 2014; The Cancer Genome Atlas, 2015). Both helical
domain and kinase domain PIK3CA hotspot mutations are strongly
activating and appear equally selectable across multiple cancer types. The
mechanism of p110a activation differs, however, with helical domain
mutations removing the requirement for binding to a p85 adapter subunit,
and therefore uncoupling PI3K from RTK activation, while kinase domain
(H1047) mutations uncouple PI3K from GTP-bound RAS (Zhao & Vogt,
2008). Although these mutations all cause constitutive PI3K activation, cells
harboring helical domain mutations might be expected to respond differ-
ently to those with kinase domain mutations when treated with drugs tar-
geting upstream regulators, such as RTK inhibitors, for instance. This
information will be useful when interpreting trial outcomes for targeted
therapies in HPVþ HNSCC and anticipating the resistance mechanisms
that may arise in the face of these therapies. A pertinent example of this is
in colorectal cancer, where kinase (exon 20) mutations but not helical
(exon 9) mutations in PIK3CA confer resistance to cetuximab, a humanized
monoclonal antibody against epidermal growth factor receptor (EGFR) that
remains the only targeted therapy so far approved for treatment of HNSCC
(De Roock et al., 2010; Markovic & Chung, 2012). Recent preclinical
studies in HNSCC cell lines and xenograft models indicate that inhibiting
OPSCC Genomics 19
PI3K or the downstream mammalian target of rapamycin (mTOR) com-

plexes in combination with EGFR inhibition could be highly effective
but it is not yet clear whether this is equally true in cells harboring helical
versus kinase domain PIK3CA mutations (D’Amato et al., 2014; Wang
et al., 2014). A further consideration when designing rational therapeutic
combinations for HPVþ HNSCC will be the role of the viral oncoproteins:
in breast cancer, for instance, preclinical data suggest that PI3Ki could be
effectively combined with CDK4/6 inhibitors to overcome PI3Ki resistance
due to downstream bypass of the G1/S checkpoint (Vora et al., 2014). This
may also be a useful strategy in HPV HNSCC but will be ineffective in
HPVþ disease, in which G1/S checkpoint is already uncoupled from
CDK4/6 activity by E7.
2.3 Copy Number Alterations and Translocations

HNSCCs display considerable genomic instability, with an average of 141
amplifications or deletions per tumor and 62 chromosomal fusions (The
Cancer Genome Atlas, 2015). Amplifications of 3q26-28, a region that con-
tains PIK3CA as well as the squamous transcription factors TP63 and SOX2,
are frequently seen in both HPV and HPVþ HNSCCs. Also common to
both HPV and HPVþ tumors are deletions on 3p, including the fragile
site gene FHIT, loss of which has recently been linked to acquisition of
the APOBEC mutational signature in lung adenocarcinoma (The Cancer
Genome Atlas, 2015; Waters, Saldivar, Amin, Schrock, & Huebner, 2014).
A number of copy number alterations (CNA) are specific to HPVþ
HNSCCs, including deletions in the tumor necrosis factor receptor-associ-
ated factor 3 (TRAF3) gene in 14% of tumors. TRAF3, which encodes a
protein involved in both innate and acquired antiviral responses, is mutated
in a further 8% of HPVþ HNSCCs, suggesting that its inactivation either by
loss or mutation is frequently selected for during the development of these
tumors (The Cancer Genome Atlas, 2015). Amplification of E2F1 also oc-
curs in HPVþ HNSCC, as does amplification of the CDKN2A locus at
9p21.3, a region that is commonly deleted in HPV HNSCC. The ampli-
fication of E2F1 indicates that in the context of pRb inhibition by E7, E2F1
protein levels may become limiting for proliferation, resulting in the emer-
gence of faster growing clones harboring amplifications. The amplification
of CDKN2A is interesting, given recent findings that p16INK4A is
required for survival of HPVþ cervical cancer cell lines through an as yet
uncharacterized mechanism involving its negative regulation of CDK4/6
(McLaughlin-Drubin, Park, & Munger, 2013). The CDKN2A amplifica-

tions seen in HPVþ HNSCC might reflect this oncogenic function.
In addition to the CDKN2A deletions and CCND1 amplifications dis-
cussed earlier, amplifications in receptor tyrosine kinase (RTK) genes,
particularly the EGFR and fibroblast growth factor receptor 1 (FGFR1),
appear to be largely confined to HPV HNSCCs (Agrawal et al., 2011;
Lechner, Frampton, et al., 2013; Stransky et al., 2011; The Cancer Genome
Atlas, 2015). RTK activation at the genetic level appears very infrequent in
HPVþ HNSCC, with the possible exception of FGFR3, which was
mutated in two TCGA tumors and fused to transforming acidic coiled
coil (TACC3) in two further tumors (The Cancer Genome Atlas, 2015).
FGFR3-TACC3 fusions were originally reported in glioblastoma
multiforme and have since been found in lung, cervical, bladder, and
nasopharyngeal carcinomas (Kim et al., 2014; The Cancer Genome Atlas
Research, 2014; Xiang et al., 2015; Yuan et al., 2014). Preliminary evidence
suggests that FGFR3-TACC3 expression confers sensitivity to the FGFR
inhibitor, JNJ-42756493 in glioma patients and to the HSP90 inhibitor
ganetespib in bladder cancer cell lines, so this translocation event, although
rare in HPVþ HNSCC could represent a therapeutic opportunity
(Acquaviva et al., 2014; Di Stefano et al., 2015).
In summary, although relatively few HPVþ HNSCCs have been
sequenced (and published data are currently restricted either to coding
regions of the genome or to targeted gene panels), thanks largely to The
Cancer Genome Atlas project we now have sufficient numbers to make
some conclusions. Clearly, certain mutations are frequently selected for
during the development of HNSCC regardless of HPV status, while other
mutations appear largely restricted to either HPVþ or HPV tumors.
This information will be important when considering stratification of
patients by HPV status for treatment with targeted therapies, as has been
attempted previously with cetuximab, for example. Differences in the
mutational mechanisms driving evolution of HPV versus HPVþ tumors,
as well as different selection pressures imposed by expression of the HPV on-
cogenes likely contribute to this diversity, further highlighting the need to
treat HPVþ HNSCC as a distinct molecular and clinical entity. It is clear
that certain activities of the HPV oncoproteins are able to phenocopy (or
at least substitute for) certain mutations during tumor development (eg,
TP53 mutations are almost never seen in HPVþ OPSCC where E6 acts
to degrade p53, whereas they are a universal feature of HPVþ OPSCC).
Only by combining our knowledge of those pathways modulated by the
OPSCC Genomics 21
HPV oncoproteins with the genomic and epigenomic data, we will be able
to predict specific vulnerabilities that may represent therapeutic targets in
these tumors. With this in mind, as research into HPVþ HNSCC moves
forward, it is essential that we continue to complement tumor “omics”
studies with a detailed examination of E6 and E7 biology.
3. THE EPIGENOME OF HUMAN PAPILLOMAVIRUS-

ASSOCIATED HEAD AND NECK CANCER
Oncogenic loci in HNSCC encompass genes that are mutated, ampli-
fied or deleted (as described above), or epigenetically compromised (epimu-
tated), for instance, by promoter methylation. Recent technological
advances have enabled the study of these epigenetic variations at the genome
level. The obtained profiles are called epigenomes and in the context of
cancer are referred to as cancer epigenomes (Lechner, Boshoff, & Beck,
2010). “Epigenome” is defined as the complete set of chemical modifica-
tions of chromatin constituents and “methylome” as the complete set of
DNA methylation modifications of the genome (Lechner et al., 2010).
Compared to the genome, the epigenome is much more dynamic,
reflecting many different functional states in time and space. These dynamics
are governed in part by reversible covalent modifications such as DNA
methylation and multiple histone tail modifications. Epigenetic processes
may affect both the host cell methylome and the viral genome itself.
One of the main mechanisms of epigenome-induced carcinogenesis is
the silencing of tumor suppressor genes, mainly mediated by site-specific
DNA hypermethylation. Such genes are heavily involved in cell cycle con-
trol, apoptosis, cell adhesion, DNA repair, and angiogenesis. Their loss of
function leads to the gain of hallmark capabilities of cancer in human cells
(Baylin, 2005). In addition to direct effects by DNA hypermethylation of
genes, various indirect effects exist. These include the silencing of effector
molecules, such as transcription factors that mainly effect downstream targets
and thereby contribute to the process of malignant transformation. An
example includes the silencing of the transcription factor RUNX3 in esoph-
ageal cancer (Long et al., 2007). The Knudson two-hit model in the context
of DNA methylation describes a model in which one allele of a tumor
suppressor gene is silenced, eg, by somatic mutation, and the second allele
is then hypermethylated, leading to a complete loss of gene function ( Jones
& Laird, 1999).
In addition to the above changes, global hypomethylation is frequently

observed in human cancers. This hypomethylation of the genome largely
affects the intergenic and intronic regions of the DNA, in particular repeat
sequences and transposable elements. It is believed to result in chromosomal
instability and increased mutation events (Wilson, Power, & Molloy, 2007).
In squamous cell head and neck cancer, it was shown to be associated with
smoking, alcohol consumption, and stage (Smith, Mydlarz, Mithani, &
Califano, 2007). DNA hypomethylation can also result in the activation
of oncogenes, thus initiating carcinogenesis. This mechanism has been
described in various cancers, eg, R-RAS and MAPSIN in gastric cancer,
S-100 in colon cancer and MAGE in melanoma (Wilson et al., 2007). In
conjunction with histone modifications (eg, the methylation at lysine
residues 9 and 27 of histone H3), these epigenetic changes affect the chro-
matin structure through a cascade of chemical reactions and interactions
with additional proteins and protein complexes.
For head and neck cancer in particular, various changes in the host
epigenome have been described. However, it has to be acknowledged
that tumor site-specific differences in epigenetic profiles exist (Lleras et al.,
2013) and this review will only focus on epigenetic changes in oropharyn-
geal cancers. Secondly, it has to be taken into account that the first few
studies in this field did not distinguish between HPV-positive and HPV-
negative head and neck cancers, in particular oropharyngeal tumors. Hence,
only a general profile of epigenetic changes was described.
With the understanding that a proportion of these tumors is caused by
high-risk HPV types and that the number of these has risen rapidly over
the last few decades, many of the published results on the epigenetic
profiling of these tumors have to be interpreted with caution.
It was suggested that these HPV-associated epigenetic changes may
explain the clinical and biological differences between these entities. More-
over, the role of epigenetic changes in early detection, metastasis, and prog-
nosis remains to be elucidated.
3.1 DNA Methylation of the Host Genome in HNSCC

For the purpose of this section only epigenetic changes tested in conjunction
with the HPV status of tumors are assessed. One of the first studies exam-
ining epigenetic changes between viral- and chemical-induced head and
neck cancers examined CpG methylation at 1505 CpG sites across 807 genes
(using the Illumina Goldengate Methylation Cancer Panel) in 68 well-
annotated HNSCC tumor samples from the University of Michigan Head
OPSCC Genomics 23
and Neck SPORE patient population were quantified. Unsupervised hier-

archical clustering based on methylation identified six distinct tumor clus-
ters, which significantly differed by age, HPV status, and 3-year survival.
Weighted linear modeling was used to identify differentially methylated
genes based on epidemiological characteristics. Methylation of CpG sites
in the CCNA1 promoter was found to be higher in HPV-positive tumors,
which was validated in an additional sample set of 128 tumors. After adjust-
ing for cancer site, stage, age, gender, alcohol consumption, and smoking
status, HPV status was found to be a significant predictor for DNA methyl-
ation at an additional 11 genes, including CASP8 and SYBL1 (Colacino
et al., 2013). In another study published in the same year, the Illumina
450k technology was applied to a set of oropharyngeal samples stratified
by HPV status (Lechner, Fenton, et al., 2013). Methylation analysis revealed
a hypermethylation signature with involvement of Cadherins of Polycomb
group target genes in HPVþ oropharyngeal cancer. Integration with inde-
pendent expression data showed strong negative correlation, especially for
the Cadherin gene family members. Combinatorial ectopic expression of
the two HPV oncogenes (E6 and E7) in an HPV-negative oropharyngeal
cancer cell line partially phenocopied the hypermethylation signature
observed in HPV-positive oropharyngeal tumors and established E6 as the
main viral effector gene. A systematic review (van Kempen, Noorlag,
et al., 2014) included 17 studies in a research analysis on methylation changes
in HPV-positive and HPV-negative head and neck cancer, including the
two above (Colacino et al., 2013; Lechner, Fenton, et al., 2013). Multiple
genes with DNA promoter methylation levels were significantly associated
with HPV status of the tumor samples in the included studies. These include
genes responsible for activation of invasion and metastasis, sustaining prolif-
erative signaling, resisting apoptosis, tumor-promoting inflammation,
evading growth suppressors, and other hallmarks of cancer (Hanahan &
Weinberg, 2011), eg, CCNA1 (Colacino et al., 2013; Weiss, Basel, Sachse,
Braeuninger, & Rudack, 2011), GRB7 (Colacino et al., 2013), SYBL1
(Colacino et al., 2013), TIMP3 (Weiss et al., 2011), SFRP4 (Marsit et al.,
2006), CDH 8 (Lechner, Fenton, et al., 2013), CDH11 (Colacino et al.,
2013), JAK3 (Colacino et al., 2013), TUSC3 (Colacino et al., 2013),
RUNX1T1 (Colacino et al., 2013), TCF21 (Weiss et al., 2011), IRX4
(Kostareli et al., 2013), GATA4 (Kostareli et al., 2013), GFRA1 (Kostareli
et al., 2013) were shown hypermethylated in HPV-positive cancers/hypo-
methylated in HPV-negative cancers and RASSF1 (Colacino et al., 2013;
Dong et al., 2003; Taioli et al., 2009; Weiss et al., 2011), STAT5a (Colacino
et al., 2013), ALDH1A2 (Kostareli et al., 2013), OSR2 (Kostareli et al.,

2013), SPDEF (Colacino et al., 2013), MGMT (Colacino et al., 2013; Taioli
et al., 2009; Weiss et al., 2011), ESR2 (Colacino et al., 2013), HSD17B12
(Colacino et al., 2013) were shown hypomethylated in HPV-positive
cancers/hypermethylated in HPV-negative cancers. The same authors later
showed that the promoter regions of TIMP3 and CADM1 are hypermethy-
lated in HPVþ OPSCC (van Kempen, van Bockel, et al., 2014). Further-
more, Schlecht et al. (2015) demonstrated that epigenetic changes at the
CDKN2A locus which is frequently lost in HPV oropharyngeal cancers
(Lechner, Frampton, et al., 2013) are associated with differential expression
of P16INK4A and P14ARF in HPVþ oropharyngeal cancer, and Chen
et al. (2015) found that methylation of IGSF4 may serve as an independent
marker of HPVþ oropharyngeal cancer. Moreover, by clustering of samples
according to CpG methylation levels, it has been suggested that a CpG island
methylator phenotype may exist for HPVþ OPSCC (Choudhury & Ghosh,
2015; Lechner, Fenton, et al., 2013).
Gene promoter methylation of some of these genes or panels of these
genes has been correlated with clinical outcome. HPV-associated promoter
methylation levels of five genes, ie, ALDH1A2, OSR2, GATA4, GFRA4,
and IRX4, were found to correlate with progression-free survival and over-
all survival in oropharyngeal cancers (Kostareli et al., 2013). Taioli et al.
(2009) reported that MGMT promoter methylation levels may serve as a
prognostic biomarker for oropharyngeal cancer, stating that promoter
methylation of the MGMT gene is significantly associated with poorer
outcome. Melchers et al. (2015) showed that DAPK1 and MGMT methyl-
ation levels have a predictive value for nodal metastasis in oral and oropha-
ryngeal squamous cell cancer. Furthermore, Notch1 methylation levels
(Colacino et al., 2013) and CDH1 (E-cadherin) methylation levels (Marsit,
Posner, McClean, & Kelsey, 2008) were suggested to be a strong predictor
for improved survival in head and neck cancer.
Similar to other human cancers, global DNA methylation changes in
oropharyngeal cancers largely affect the intergenic and intronic regions of
the DNA (Furniss, Marsit, Houseman, Eddy, & Kelsey, 2008; Poage
et al., 2011; Richards et al., 2009; Sartor et al., 2011). Long interspersed
repeat sequences (LINE-1) were found to be significantly hypomethylated
in HPV head and neck cancers in three studies. Richards et al.
(2009) showed a significant correlation of HPV status and normal LINE-1
methylation levels in 26 HNSCC samples. Sartor et al. (2011) included 49
oral cavity and oropharynx SCC samples in his analysis and reported that
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is of course extremely hard, but there are other hard woods like
cedar, and many varieties of valuable timber.
Yerba mate is cultivated in many places, especially in Misiones;
the wild growing trees of the forest furnish a still greater supply of the
leaves. From these a drink is made which outside of the large cities
is in this part of the continent far more popular than tea or coffee.
The northern forests contain several varieties of rubber trees, but
none are exploited. Along the Andes are forests, the principal ones
from Lake Nahuel Huapi south. Those in this region are believed to
be worth $10,000,000,000. The variety of native woods both hard
and soft is large; and trees of other countries have been introduced.
Thousands of eucalyptus trees have been planted on many
estancias, serving a useful purpose in many ways, beside being an
ornament on the level plain.
Mining
In the description of the Provinces, mention has been made of the

minerals existing in various localities, but up to the present time the
working of these has been slight. Tungsten, gold, copper, wolfram,
borax, and petroleum have received the most attention, but few are
those who have realized any considerable profits. Within ten years
the exports have amounted to hardly more than $3,000,000.
Gold is mined in small quantities in various places; in southern
Patagonia it is gathered from the coast sands after a heavy sea
storm. In Neuquen and Catamarca are workings of fair size. The
copper deposits of the Andes are difficult of access but may be
developed later. Silver was mined formerly, but the ore was of
moderate grade and the work was discontinued. A reverberatory
smelter has recently been installed for the mines in Rioja.
Coal deposits exist in Mendoza, San Juan, Neuquen, Chubut, and
Tierra del Fuego. The coal is not very good but will help in view of
the shortage and high prices. Work is being done in San Juan and
Mendoza. It is proposed to open mines among large deposits in
Chubut, though the coal will have to be carried 180 miles to a
railway.
The tungsten industry is active; 900 tons have been mined in one
year, about one-seventh of the world production. Exploitation of
marble, wolfram, and mica in Córdoba and San Luis is showing good
results.
Manganese is exported in increasing quantity chiefly from the
desert section of Santiago del Estero.
Large saline deposits exist, some in basins with no outlet, in the
central Provinces, some of volcanic type on the Puna of Atacama,
others near the ocean not far from Bahia Blanca. Some of them have
been exploited. Importation of salt has diminished and home
production is expected shortly to suffice for local needs.
Petroleum is now exciting the greatest interest. Oil is known to
exist in four regions with others reported, but only one has been
thoroughly tested, that at Comodoro Rivadavia. Borings carried on
here by the Government struck oil at a depth of about 1800 feet in
1907. In 1910, 12,000 acres were reserved for Government
exploitation which has since been carried on. The place is near the
coast about 850 miles south of Buenos Aires. Development has
been rather slow, but in 1916, 25 wells had been sunk and 21 were
in active production of about 14 tons each per day. There were four
steel tanks and other storage space, in all about 26,000 cubic
meters, one such of this oil equalling .93 metric ton. In 1917, 36
wells were in production and 19 being bored. In 1918 about
1,250,000 barrels were produced. Tank steamers are provided, and
storage tanks in Buenos Aires. An oil tank is begun in Rosario.
Others are to be constructed in Buenos Aires, Bahia Blanca, Puerto
Militar, Santa Fé, and Mar del Plata. The oil is heavy with an asphalt
base; distilled, it yields 1.5-3.5 per cent of naphtha and gasoline, 15-
19 per cent of illuminating oils, and 77-85 per cent of lubricating oils,
fuel, and coke. Heavier than the better grades of United States oil, it
has been used almost entirely as fuel, though it is said that it will
distil readily. This will undoubtedly be its chief usefulness, to serve
instead of coal. It is employed by a number of factories. A new
Government well, 1921, was producing 34,000 barrels a day, and
prospects are of the best. Millions have been appropriated for tank
steamers, machinery, and for intensive development of the oil fields.
The price rose from about $10 a ton in 1916 to $40 in December,
1917. Government control will probably continue, especially because
the oil is likely to be used by the navy.
A few private companies are operating outside the restricted area,
using 12-inch tubes, while the Government has used smaller. One
Company with a capital of $2,000,000 has with other equipment 4.3
miles of railway connecting with the Government railway to the port,
also two miles of pipe line. Many of the frigorificos use oil, mostly
Mexican. The West India Oil Company imports from the United
States or Mexico, mainly for refining.
The other fields are the Salta-Jujuy, the Cacheuta, a few miles
south of Mendoza, and the Mendoza-Neuquen field, 700 miles
southwest of Buenos Aires. In these fields the oil has a paraffine
base, a sample from Jujuy showing 5 per cent of light oil, 30 per cent
of kerosene, and 52 of lubricating oil; a grade equal to that of
Pennsylvania or Ohio. One such field in Neuquen justifying
immediate development is favorably located 824 miles from Buenos
Aires near the Ferrocarril del Sud, which will provide special cars
and tariff, so that speedy results are hoped for.
Industries including Manufactures
While Argentina is primarily an agricultural and pastoral country

and is likely so to continue, a fair amount of capital is invested in
manufacturing and in other commercial projects, some of the capital
European. The largest sum is invested in Light and Power
Companies; for all cities of any size have electric lighting and many,
electric traction. About $128,000,000 have been thus invested; in
packing houses 40 millions, flour mills 7; in sugar refineries 50, wine
making 78, foundries and metal works 25, dairies, etc., 43, tannin
extract, etc., 33, lithographing and printing 12, breweries 14,
construction companies 11, these all millions. Other companies with
investments of 5-10 millions are shoes, saw mills, jute and cotton
sacks, carpentry, painting and horse-shoeing together; liquors,
tanneries, cotton and woolen mills, furniture, trunks and tapestries,
leather goods, brick kilns, clothing, wagons and carriages, ice and
aërated waters, ore smelting, grain elevators; many more with
investment of 1-5 millions, besides a total of 47 million in still smaller
companies.
Of other lines, in 1916 there were 71 telephone companies, capital
$15,000,000; 143 banks, capital about $500,000,000, employing
10,000 persons; 85 insurance companies. The companies of all
kinds generally are on a sound and paying basis, in spite of high cost
of fuel and power. The two leading department stores in Buenos
Aires, equal to our best in character, are both British owned; there
are many other shops of every kind of the highest class.
The tramways and subway of Buenos Aires are noteworthy. The
tramway system has been called the best in the world. There are
500 miles of lines, carrying about 400,000,000 passengers yearly.
Packing like cattle is not allowed, the number of persons per car
being strictly limited. The subway 8.7 miles long carries 2,500,000
with a splendid system and fine service including one for freight.
Extensions were postponed on account of the War. The water
supply, taken from the river, and the drainage system are of the
highest character. A revenue of $7,000,000 is received by the
Government.
Dairy Products. The dairy industry is of quite recent development;
butter formerly imported in quantity is now becoming an article of
export. From 1914 to 1918 the production of cheese increased 277
per cent, of butter 162 per cent. Three thousand existing creameries,
most of which are in the Provinces near the Capital, in 1918 made
50,000,000 pounds of butter, 40 million of cheese, and 15 of casein.
More than half the butter and some cheese was exported.
The growth of other industries has caused a great falling off in
many imports; of preserves 62 per cent, of ham 96, preserved
vegetables 87, beer 98, cider 55, common wine 85. Other things as
chocolate, macaroni, fruit crackers, shoes, and cloth have decreased
50 per cent. Of fideos enough is produced for home use, and to
export in 1918 over 2,000,000 pounds.
Furniture. The construction of furniture has attained large
proportions, a great part of what is sold in the country being made in
Buenos Aires though often bearing foreign names. Wood is
imported, even $25,000,000 worth in one year, while the finest
woods grow in the country. Of 305 saw mills, 134, the most
important, are in the City and Province of Buenos Aires far from the
forests. The small mills near the woods merely chop off the branches
for transport or prepare firewood. Cut wood from Buenos Aires is
returned to Misiones and Corrientes for construction, a terrible
waste. A change has begun; the mills near the forests are being
enlarged and equipped with machinery, so an evolution of the
industry is under way.
Paper. Eight paper factories with capital of $8,000,000 employing
1500 persons produce 40,000,000 pounds of paper, 28,000,000 for
packing, the rest for newspapers, books, and other things. A great
quantity is still imported, formerly from Germany, lately much from
the United States. The paper is made of rags, shreds of paper, and
pasteboard, the consumption of pulp being small, hardly 200,000
pounds. One factory at Barranqueras, on the Paraná River in the
Chaco, employs a kind of bog grass to make three tons of straw
board a day.
Flour. The flour mills are of great importance, supplying in 1919,
850,000 tons of flour for home use and some for export. In 1918,
176,445 tons were exported. With fewer mills than formerly, the 400
existing are more productive. The 79 in Buenos Aires, 47 in Santa
Fé, 44 in Entre Rios, and 26 in Córdoba produce 95 per cent of the
total. Sixty-one per cent of the mills are Argentine owned. One
hundred and fifty two are steam mills, 156 hydraulic. They have
25,000-30,000 horse power and employ 10,000 persons. About
$34,000,000 are invested in the industry while the production is
$100,000,000. However the farmers have trouble, as the fee for
hauling grain has increased 60 per cent, and cartage 25 miles to a
station is as much as the freight from Buenos Aires to New York. A
flour mill in Mendoza and in other western cities of the wheat belt
would undoubtedly pay handsomely, saving expensive transport. A
new flour mill at La Plata to cost $500,000 is to turn out a quantity
sufficient to fill 1000 bags a day.
Beer is made in 25 factories for the consumption of the entire
country, a quantity of 80,000,000 litres worth $12,000,000. To
produce the 7000 horse power needed, thousands of tons of coal,
wood, and petroleum are consumed.
Other Manufactures. Factories making shoes, said to be of the
best quality, underwear, umbrellas, acids, perfume, and many other
articles are found. Vegetable oils are extracted from peanuts,
linseed, rape, cotton seed, and other articles, in establishments in
Buenos Aires and Santa Fé.
Altogether there are about 50,000 industrial establishments with a
capital of $800,000,000 using 678,000 horse power, employing
500,000 persons, consuming nearly $500,000,000 worth of material,
and producing nearly $1,000,000,000 worth of goods. About half of
these are extractive or manufacturing. One-third belong to the
Argentines who supply 18.67 per cent of the capital.
Developing Industries. The Government is interested in the
establishment of other factories and construction work; a cement
factory in Buenos Aires to make 300,000 tons per annum is
considered, the Government now using 700,000 tons a year. Ship
building is encouraged; a steel ship of 1250 tons was launched at
Riachuelo; yards are to be constructed at the port of Carmen de
Patagones on the Rio Negro by an Argentine company with capital of
50,000,000 pesos. Some armored cement oil-tanks of 6000 tons
capacity are to be made for Comodoro Rivadavia, and a depot for
petroleum and naphtha at the port of Mar del Plata. Also for the
former, port works, a breakwater, a mole for loading, and houses for
workmen at a cost of 17,000,000 pesos. Sanitary works for 16 towns
at a cost of 9,800,000 pesos are provided for, 22,000,000 pesos are
to be spent in three years for machinery and tank steamers to
develop the Government oil wells, the exploitation of which will cost
45,000,000 pesos; present production is yet insufficient.
A Spanish Argentine Corporation with a capital of $10,000,000 is
to build two large frigorificos at Buenos Aires and Santa Fé, with
steamers to transport beef to Spain. Another frigorifico is designed
for Puerto Deseado in the south to coöperate with local ranchmen.
The lack of combustibles has for many years been a great and
embarrassing problem, an early solution of which is now hoped for.
Importation of coal from England and of petroleum from the United
States has been carried on at great cost. In five years, 1912-16,
$190,000,000 was spent for such articles, while as they say
petroleum ran into the sea and wood rotted at the railway stations. In
1919 coal was $26 a ton. The forests of the north have an
inexhaustible supply of wood; the charcoal industry is quite well
developed in the Chaco, north Santa Fé, Tucumán, and Santiago.
Many woods are appropriate, but high freights have impeded their
use. A large deposit of coal is recently reported in Tucumán. A new
railway to the firewood region of Santiago del Estero will save a 100
mile haul. A splendid source of electric power are the Iguassú Falls
with 275 cascades, the greatest with a height of 213 feet.
Investigation shows that 500,000 horse power is easily available,
one half each for Brazil and Argentina. Ten thousand horse power
would be sent 800 miles to Buenos Aires, the rest used in Misiones,
Corrientes, and Entre Rios. From the Salto Grande Falls on the
Uruguay 50,000 horse power might be available for Argentina and
Uruguay each.
Investments
The opportunity for investments for persons with capital is

evidently large. A great deal of money is necessary for stock raising,
though less in the far south. Agriculture, especially the raising of
cotton, rice, or tobacco might be attractive; the sugar industry may
be extended. Factories of various kind may be established by
experts. Persons speaking Spanish fluently, capable of acting as
foremen or superintendents of establishments of various kinds, might
find employment. The minerals with the exception of petroleum are
less accessible than in some other countries. An American
department store in Buenos Aires is desirable, and one might
succeed in Rosario.
CHAPTER XXXVIII
PARAGUAY: AREA, HISTORY, GOVERNMENT, POPULATION, ETC.
Paraguay has been called the most romantic of all the South
American countries, from the point of view of nature and history both.
It is a land of “dolce far niente” so far as agreeableness is
concerned, a land where nature is lavish and necessities are few; on
the other hand a region where the climate is not enervating, where
energetic action and enterprise are not altogether lacking, and where
these find ample reward. One of the two inland countries of the
continent, having always been such, she has no grievance on this
account. In fact, being in the heart of South America and almost
surrounded by rivers, Paraguay has in many ways a most favorable
location for inland commerce, which will surely bring her prosperity.
Area, Population, Boundary
Area. Paraguay, generally called eighth in size of the South

American Republics, has an area of about 165,000 square miles,
more or less, according to the determination of the boundary dispute
with Bolivia. One hundred and ninety-six thousand miles is claimed
by the Paraguay Government. The more settled part of the country
east of the Paraguay River covers about 65,000 square miles; the
section west, which on most maps is given to Paraguay, though
claimed by Bolivia, is more uncertain in area, but may have 100,000
or more square miles. With the other neighboring countries the
boundaries have been definitely determined.
Population. As in several other countries, the population of
Paraguay can be estimated only, since no exact census has ever
been taken. Approximate figures given by different writers are
800,000-1,100,000.
Boundary. Paraguay has Brazil on the north and east; Argentina,
too, is east, is exclusively on the south, and partly on the west; while
Bolivia is west and north of the Chaco, the dividing line being
uncertain.
The greater part of the way rivers form the boundary line. The
Paraná separates Paraguay proper from Argentina on the south, and
on the east as far as the Iguassú River; north of the latter river Brazil
is on the opposite shore of the Paraná to beyond the Sete Quedas,
or Falls of La Guayra (higher up the Paraná is wholly in Brazil); the
boundary line then goes west and north along the watershed to the
source of a branch of the Rio Apá, which with that river it follows
west down to the Paraguay. Above the point of junction the Paraguay
River has Brazil on the east bank and for some distance the
Paraguayan Chaco on the west. Below the Apá, the Paraguay
divides the country into sections, as far as the Pilcomayo River, on
the southwest of which is Argentina. Below, along the Paraguay
River to the Alto Paraná, Argentina is on the west. The Chaco
section may be said at present to be in the practical possession of
Paraguay, so far as it is occupied by any one except savages.
History
It seems extraordinary that here in the centre of the South

American continent, 1000 miles from the sea, is one of the oldest
cities, the capital of the Republic, Asunción, founded a full century
earlier than Providence (1636), and 85 years before the landing of
the Pilgrims at Plymouth. Juan de Ayolas, sent by Pedro de
Mendoza after the first settlement at Buenos Aires (not long after
destroyed), in 1536 established here a colony; then sailing up the
river to a place he called Candelaria, with a few followers he boldly
struck off through the forest towards Peru. Irala, left behind to await
his return, proved faithful; but after long waiting in vain, being obliged
twice to go to Asunción for provisions, he learned at last that Ayolas,
after a successful journey to Alto Peru, had arrived in his absence
and had then been slaughtered by savages: a tragic sequel of
heroism, fitly to be compared to that of Captain Scott, his
achievement of the South Pole, followed by the disastrous return
journey.
Irala, later becoming Governor, was one of the few
Conquistadores, after many vicissitudes, to die peacefully in 1557.
Until 1776, Asunción was the chief Spanish city of the eastern slope
of South America. Buenos Aires then became the seat of a Viceroy,
in that year appointed.
In 1810, after expelling the Viceroy, the Argentines sent up a small
army, expecting Paraguay also to revolt, but she declined. However,
in 1811 the Paraguayans expelled the Spanish Governor; in 1814 a
despotism under Dr. J. G. R. Francia came into being. At his death in
1840, his nephew, Carlos Antonio Lopez, succeeded to power,
followed in 1862 by his son, Francisco Lopez. This young man, who
had visited Europe, soon organized a well drilled army with the idea
of becoming a second Napoleon, in South America. His opportunity
came when Uruguay appealed for his assistance against the
Portuguese of Brazil. Crossing Argentine territory to invade Brazil, in
spite of the refusal of Argentina to give permission, Lopez became
involved in war with both of the larger countries and later with
Uruguay. Although so small, Paraguay might have held her own
against one of the larger Republics, but after making a desperate
struggle for nearly six years, during which most of the male citizens
were killed, boys of 12-15 forced to fight, women compelled to work
in the fields and to act as supply bearers, with cruel persecution from
the Dictator, the army was utterly wiped out, and Lopez killed in
1870. Three-fourths of the population had perished.
Not yet has the country recovered its previous condition and
numbers, several revolutions having interrupted its growth; the last in
1911-12. Curiously, the people pride themselves on being one of the
most homogeneous of the South American Republics and the best
fighters.
Government
The Government organized in 1870 is of the usual form with three

branches. The President, elected for four years, is not again eligible
for eight years. There are two Houses of Congress, a Senate of 13
members and a Chamber of 26 Deputies. A Permanent Committee
of Congress sits during the interval between sessions, both to
provide for emergencies and to prevent usurpation by the Executive.
The Judiciary has a Supreme Court, two Superior Courts of Appeal,
Civil and Criminal Courts with Lower Courts and Justices.
The section west of the River is under military command; the part
east has 23 electoral districts with subdivisions. As in Argentina, all
persons born in the Republic are deemed citizens.
There are said to be 20 Districts, some of which along the
Paraguay from the north are Concepción, San Pedro, Villeta, Pilar;
farther east Caraguatay, Yhú, Paraguarí, Guaira, Guindí, Caazapá,
San Ignacio, and Encarnación. All have capitals of the same name
except Guaira, the capital of which is Villa Rica. Data as to area and
population of these is lacking.
An unusual official is a Defender General of the Poor, of Minors,
and of Absentees.
Population
The Paraguayans claim that their people are nearly pure Spanish,
with slight admixture of Guaraní, this being the most numerous,
intelligent, and peaceful of the Indian tribes of that region. Preserving
the spirit of the invaders, they are an unusually hardy race for one on
the edge of the tropics. While Spanish blood and culture largely
predominate in the capital, the population of the rural districts is
more primitive in character.
The number of the people is uncertain, but may reach 1,000,000,
including 50,000, some say 100,000 wild Indians belonging to
several tribes, chiefly in the Chaco. In the eastern section of
Paraguay proper there are probably not over 25,000 persons, most
of the population being east and south of the mountainous section
and especially near the Paraguay River. They say that there are no
idle or poor in the country, although many may be seen barefoot in
Asunción; as the simple life is popular, little clothing is needed, and
food is abundant. (Of late the standard of living has been rising.) For
this reason the country people generally lack energy and ambition.
Most of the inhabitants are engaged in agriculture, some also in
pastoral pursuits. The native women are called superior to the men.
Burdens thrown upon them during their great war made them
resourceful and independent. The men as a rule are peaceful unless
they have been drinking caña, which is now forbidden by some large
employers of labor. In the section east of the mountains are
uncivilized Indians of the principal tribes, one timid and retiring.
Some of the Cainguaes at times work in the yerbales. The Indians in
the Chaco are of a number of different tribes of a low order of
civilization, who are mostly nomads, and live by the chase and
fishing. A few work at estancias spasmodically, never for long.
Education
Education is backward, though the percentage of illiteracy, if about

60 as is said, may be less than in some of the other Republics. With
the scattered population, chiefly in the country and in small towns, to
provide educational facilities for all is impossible. There is a
University in Asunción with five Faculties; secondary schools in
Asunción, Pilar, and Villa Rica; and six Normal Schools. A School of
Commerce with night and day classes does good work in the capital,
where the Instituto Paraguayo is of important cultural value, recently
receiving a library of 2000 volumes in English from the Carnegie
Peace Foundation. The Banco Agricola has done much for the
diffusion of general education as well as more specifically in
agriculture. As in other countries, some young men go abroad to
study, either on Government scholarships or with private means.
Press, Religion, etc.
Press. The capital is well supplied with newspapers and

periodicals; little journalism exists elsewhere.
Religion. The religion is Roman Catholic, but there is complete
toleration for other creeds. An important work has been carried on in
the Chaco by W. Barbrooke Grubb and others of the English Church.
Schools have been established, with an industrial and intellectual as
well as a religious centre. Civil marriage alone is legal.
Telegraphic communication with the outside world is carried on by
way of Posadas and Corrientes, but frequent interruptions in service
have made it unsatisfactory. There are over 2000 miles of wire in the
interior. Wireless is used by the Government to communicate with
the garrison towns. There are stations at the Capital, Concepción,
and Encarnación. In 1919 there was no Telephone service in
Asunción, as the station had been destroyed by fire; but such service
existed in Concepción, Villa Rica, and Paraguarí.
Money. The Money in use is paper, the standard, a gold peso
equal to the Argentine, 96.5 cents. The paper peso varies in value, in
1920 being worth five cents. The value of exports and imports is
declared in gold, an Exchange Bureau supplying local money at the
actual rate. Thus fluctuations affecting commerce have ceased and
business has improved.
The Metric System of weights and measures is usual.
CHAPTER XXXIX
PARAGUAY: PHYSICAL CHARACTERISTICS
Highlands. The physical features of the country are simple, the

Paraguay River dividing it into sections: the Gran Chaco, to some
extent unexplored in detail, and the Oriental. The latter, Paraguay
proper, is traversed from north to south by a broad irregular belt of
highlands, nowhere much above 2200 feet. The angle of the ridges
is sharper on the west, the country undulating in gradual slopes from
the foot. On the east, spurs run out enclosing deep valleys, and the
country has more of an upland character. The streams flowing west
to the Paraguay are smoother and to some extent navigable, while
those flowing east and southeast to the Alto Paraná are interrupted
by rapids and falls.
Plains. In the southwest section between the Paraguay and the
Paraná there is considerable marshy country, part draining into the
Ypoa lagoon; other marshy lowlands occur along the Paraguay
River. All these could be drained at moderate expense, thus
providing much rich and accessible land. The western section,
containing most of the inhabitants, has an average altitude of about
600 feet. In the more settled parts of the west and south are
extensive grassy and open lands, and hills covered with forests,
while clumps of trees are frequent in the lowlands. The soil of the
western part is rather dry and sandy except near the rivers and
marshes. It is extremely rich, of a reddish color due to impregnated
iron. Above is a thick layer of humus, formed by centuries of
decaying vegetation. In places the soil is clayey, or has a substratum
of clay beneath. This is true of most of the forest region, and of the
swampy section. Of Paraguay proper the western part is 25 per cent
forest, the middle 66, the eastern 95 per cent.
The Gran Chaco, though called a plain, and flat near the rivers, is
somewhat hilly. Rarely, a freshet on the Paraguay or the Pilcomayo
transforms the neighboring country into great lakes, these
sometimes extending 20 or 30 miles back from the river. Along the
river banks is usually a narrow strip of forest, from which the soil and
trees occasionally crumble into the stream, especially along the
Pilcomayo, thus impeding navigation on that river. Farther back are
open plains dotted with groups of palms, and some stretches of
forest. The average altitude is 426 feet. Little is known of the country
north of 22°.
Rivers. The country is greatly favored with rivers. The Paraguay,
1800 miles long, rises in Matto Grosso, Brazil, near the source of the
Tapajós, a branch of the Amazon. From the east the Paraguay
receives several important streams. Below the Apá, the northern
boundary, two tributaries, the Aquidabán and the Ipané, are nearly
200 miles long. More important is the navigable Jejui (all these north
of Asunción); still more the Tebicuary, entering the Paraguay far
south of Asunción by two mouths, one over one-half, the other over
one-third of a mile in width. The Alto Paraná is different, a more
rocky and a shallower stream than the Paraguay. From that country
it has many affluents, the Monday over 100 miles long with great
cataracts in the lower part but navigable above. The Paraná River,
2000 miles long to the mouth of the Uruguay, rises in Goyaz, 665
miles above the Sete Quedas. It is 100 miles more to the Iguassú,
then 492 to Corrientes, and 676 beyond to the beginning of the La
Plata.
The rivers of the Chaco are more sluggish. Even the Pilcomayo is
untraced in its middle course where vast swamps impede passage
by land or water. The river is navigable in its lower reaches for a
considerable distance, and the upper part is well known. The River
Confuso is a smaller stream of similar character. Paraguay has few
lakes save those swampy in character, but one, Lake Ipacaraí, east
of Asunción, is a popular beauty spot, with pleasure and health
resorts. Lake Ypoa is larger and both are navigable by boats of slight
draft.
Climate
The climate of the country is called ideal, that of course depending

on one’s taste. In general it is subtropical with two seasons, the
summer temperature averaging 81°, the winter 71° or less. The
rainfall is fairly distributed, the most in the hot months, December to
February. Sudden changes of weather occur; from hot, humid north
winds, or cooling south winds from the Argentine plains. In places
the winter temperature may fall to 33°-42°, even with frost, but not
near the Paraná River where there are fogs. In summer the
maximum temperature at Asunción occasionally reaches 100° or
more, and there are 60 inches of rain; more farther east, but less in
the Chaco.
CHAPTER XL
PARAGUAY: THE CAPITAL AND OTHER CITIES
The Capital
Asunción, the population of which is variously given as 80, 90,

100, 120, and 125 thousand, is a quiet town on the Paraguay at a
point where the bank rises to a considerable height, affording good
drainage, and from the palace a pleasing view. An English writer
calls the city the nicest, cleanest town above Buenos Aires. Although
1000 miles from the ocean, it has an altitude of but 203 feet above
sea level. The classical building of the Custom House stands by the
river side. The palace above, built by the Dictator, Francisco Lopez,
as a residence, is used for the offices of the President and his
Cabinet. Other important buildings are a House of Congress, a
Cathedral, a Museum of Fine Arts containing a Murillo, and a
National Library with many priceless documents.
The several hotels are fairly comfortable if not exactly modern in
character. There are electric lights and electric car service. The
streets at last accounts were poorly paved with rough cobble stones,
but some automobiles are in use. The city is steadily advancing in
commercial importance, 80 per cent of the imports and 40 of the
exports passing through its Custom House.
Other Cities
From the meagre information available, to describe individually the

various States or Districts of Paraguay is impossible, as also
unimportant, the sparse settlements having little marked variation.
Mention will be made instead of the principal towns, few of which
have a population of 15,000.
Villa Rica, population perhaps 30,000, 40 hours from Buenos
Aires and 93 miles from Asunción, is the second city of Paraguay.
Situated in a rich agricultural district, it is destined to a sound if not
rapid development. It will become important later as a railway
junction; for the railway designed to cross the Paraná River into
Brazil just above the entrance of the Iguassú, passing the great
Falls, will connect with the São Paulo-Rio Grande Railway at União
de Victoria and go on to São Francisco, called the best port in Brazil
south of Santos. However, the date of this road’s completion is
uncertain. The actual railway junction is at Borja, a little south of Villa
Rica, but the latter place will receive the benefit. All of the towns are
what we might call large country villages.
Villa Concepción, 130 miles up the river from Asunción, the third
if not the second city of commercial importance, with a population
given as 16, 25, also 30 thousand, is a port for yerba, cattle,
quebracho, and sugar. The town of one story houses has better
streets than those of the Capital, and a comfortable inn. It may now
boast of 50 automobiles, instead of the one stylish turnout noted
years ago by Colonel Roosevelt.
Villa Encarnación, population 15,000 or less, opposite Posadas,
is another city of commercial importance, as the junction of the
railway ferry and of river transportation. Here there is a change of
river boats, those of lighter draft being required for the Upper
Paraná.
Other towns are Paraguarí on the railway between Asunción and
Villa Rica, Carapeguá near by, San Pedro north of Asunción, half
way to Concepción, Luque, nine miles south of the Capital, and Pilar,
well down the river. Villa Hayes is one of several centres of
commercial importance in the Chaco.
San Bernadino on Lake Ipacaraí, two hours from Asunción, is a
pleasure resort favored in winter by citizens of Buenos Aires. A large
modern hotel caters both to fashionables and to sufferers from
tuberculosis.
Transportation
River transportation in Paraguay is of the greatest importance.

Large comfortable steamers give good service three times a week
between Buenos Aires and Asunción, making the trip upward in four
or five days, down stream in three. Above Asunción boats of suitable
size and draft give poorer service on the Paraguay. Several lines run
up 765 miles to Corumbá in Brazil, a three or four days journey,
calling on the way at several Paraguayan ports, Concepción, 250
miles, San Salvador, Puerto Max, and others. Fares are high,
especially down stream, apparently because one is obliged to go.
Some ports on the Paraguay and on a few branches above and
below the capital are visited by local steamers. Barges can be
floated on over 2000 miles of internal rivers. Chatas (which are poled
along) drawing 2-4 feet of water carry 10-100 tons; such boats on
the Paraguay may carry 600 tons.
On the Alto Paraná, as previously stated, there is semi-weekly
service from Corrientes to Encarnación and Posadas. Above, three
times a week steamers, with many calls on both sides of the river,
make a four days’ journey to the Iguassú and beyond to Puerto
Mendez, from which point a short railway in Brazil passes around La
Guayra Falls.
Railway facilities are increasing, construction though not so easy
as on the Argentine plains being less difficult than in general in the
other Republics. Nearly 475 miles of track are in operation, 200 of
them privately owned. The main line of the Central Paraguay,
Encarnación to Asunción, 230 miles, has first class equipment with
through sleepers to Buenos Aires, 966 miles from Asunción. Several
short lines of very narrow gauge, about 30 inches, transport
quebracho and timber from the interior to river ports, 152 miles of
these in the Chaco. A railway of 40 miles serves sugar mills back of
Concepción. If the cross lines proposed to Brazil and Bolivia (of one
of the former some miles are in operation) are constructed within the
decade, a rapid development will ensue. Cart roads are few and very
poor. In wet weather six or eight oxen are required for a cart. Two-
wheeled vehicles are much employed.

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The Genomics, Epigenomics,

Advances in Genetics, Volume 93

6. Linking the Cancer-Associated ChangesdA Model of Human Papillomavirus- 38

However, a proportion of HNSCC patients (up to 20%) have little or no

1.1 The Role of Human Papillomavirus in Head and Neck

M. Lechner and T.R. Fenton

1.2 Human Papillomavirus-Driven Carcinogenesis

immune-privileged lymphoid tissue (Egawa et al., 2015). Development of

Figure 2 Interaction of the HPV16 E6 and E7 oncoproteins with multiple cellular

2. THE GENOME OF HUMAN PAPILLOMAVIRUS-

transformation requires the acquisition of lesions in the host genome, hence

2.1 Mutational Processes Shaping the HPVþ HNSCC

uncover a number of mutagenic processes at play during tumor develop-

2.2 Recurrent Mutations in HPVþ HNSCC and

for Wnt pathway mutations during HNSCC development. Wnt signaling

PI3K or the downstream mammalian target of rapamycin (mTOR) com-

2.3 Copy Number Alterations and Translocations

(McLaughlin-Drubin, Park, & Munger, 2013). The CDKN2A ampliﬁca-

3. THE EPIGENOME OF HUMAN PAPILLOMAVIRUS-

In addition to the above changes, global hypomethylation is frequently

3.1 DNA Methylation of the Host Genome in HNSCC

and Neck SPORE patient population were quantiﬁed. Unsupervised hier-

et al., 2013), ALDH1A2 (Kostareli et al., 2013), OSR2 (Kostareli et al.,

In the description of the Provinces, mention has been made of the

Industries including Manufactures

While Argentina is primarily an agricultural and pastoral country

The opportunity for investments for persons with capital is

Area, Population, Boundary

Area. Paraguay, generally called eighth in size of the South

It seems extraordinary that here in the centre of the South

The Government organized in 1870 is of the usual form with three

Education is backward, though the percentage of illiteracy, if about

Press, Religion, etc.

Press. The capital is well supplied with newspapers and

Highlands. The physical features of the country are simple, the

The climate of the country is called ideal, that of course depending

Asunción, the population of which is variously given as 80, 90,

From the meagre information available, to describe individually the

River transportation in Paraguay is of the greatest importance.

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