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Supplemental Information
Andrew P. Stein, BA1,2, Sandeep Saha, MS3, Jennifer L. Kraninger, BS2, Adam D. Swick, PhD2, Menggang Yu, PhD3,
Paul F. Lambert, PhD*,1, and Randall J. Kimple, MD PhD*,2
Departments of Oncology1, Human Oncology2 and Biostatistics3,
University of Wisconsin School of Medicine and Public Health
Madison, WI 53706, U.S.A.
1
Supplemental Methods
We followed the PRISMA statement guidelines throughout all elements of our analysis and manuscript preparation.23
Article selection
A systematic literature review was performed using the NIH PubMed search engine to identify articles published
through January 31, 2014. We utilized keyword search terms including HPV, oropharyngeal, cancer, tonsillar, and
prevalence. Specific search phrases employed were “HPV prevalence in oropharyngeal cancer”, "HPV and tonsillar
cancer" and "HPV prevalence in tonsillar cancer". Titles and abstracts of all studies displayed by these broad searches
were examined to identify any articles that presented data on the prevalence of HPV in OPSCC by PCR, in situ
hybridization (ISH), p16 immunohistochemistry (IHC), or other molecular methods. Studies that met these initial criteria
were selected for detailed evaluation of the entire study. Reviewing the reference sections of this initial cohort of articles
identified additional relevant papers missed by our PubMed searches. These newly identified articles were subjected to the
same process described above whereby the titles and abstracts were examined first, and a portion of the studies were
subsequently selected for full review.
Exclusion criteria
Articles were excluded if they were reviews, did not utilize molecular techniques to determine HPV status, failed
to distinguish oropharyngeal cancers from other regions of the head and neck, or were not written in English. To be
included in our meta-analysis, all articles needed to present the first and last years that their samples derived from (sample
collection period), number of HPV-positive OPSCCs, total number of OPSCCs analyzed, and the country/region where
the samples originated. Eighteen articles did not report the sample collection period and three failed to present the number
of HPV-positive OPSCCs detected. Before excluding these articles, we contacted the authors to gather the missing
information. We received the queried data from 14 of 18 authors related to the sample collection period and two of three
regarding the number of HPV-positive OPSCCs detected in their study. If we did not receive the necessary information
from the authors, these papers were excluded. Finally, we carefully examined all articles in order to exclude ones with
overlapping patient populations (we included the article reporting on the larger number of patients) (Supplemental Table
S1). The ultimate decisions for article inclusion/exclusion were made jointly between two of the authors (APS and RJK).
Data abstraction and organization
The following data was collected from all articles meeting our inclusion criteria: authors, journal, publication
year, country, description of patients, anatomical sites of cancers analyzed, sample preservation technique, HPV detection
2
method, sample collection period, overall number of head and neck cancers analyzed, number of cancers that were HPVpositive and breakdown of HPV subtypes (if documented). Some studies employed multiple methods for HPV detection.
In these instances we collected data based on the method the article utilized as their standard for evaluating HPV status.
To focus our analysis on the prevalence of HPV in OPSCC, we stratified the head and neck cancers reported by
each study into oropharyngeal (tonsil, base of tongue, soft palate, and lateral/posterior pharyngeal walls) and nonoropharyngeal (all other sites) subgroups. Our final data included the overall number of HPV-positive OPSCCs out of the
total number of OPSCCs analyzed by a specific article. Detailed data extracted from each manuscript is documented in
Supplemental Table S2, and the additional information gathered from the authors we contacted is described in the
footnotes of this table.
Based on the country of origin for the samples analyzed, each study was separated into one of six distinct
geographical regions: North America, Europe, Asia, Australia, South America and International. Due to the limited
number of articles from Asia (n=11), Australia (n=2), South America (n=2) and International (n=3), we combined these
studies into a single category labeled “Other”. To assess changes in the prevalence of HPV-positive OPSCCs over time,
we used the median year of each article’s sample collection period (rounded down to the nearest year) to separate articles
into four discrete time frames: pre-1995, 1995—1999, 2000—2004 and 2005—present. As shown in Supplemental Table
S2, 13 articles separated their samples into multiple collection periods. Therefore, we had a total of 135 data points from
the 105 articles.
Statistical analysis
Due to the small number of articles for individual countries, we could not carry out appropriate statistical analyses
examining trends over time for each nation (aside from the United States24). Therefore, we examined broader regions and
determined if HPV prevalence was static or changed over time worldwide, in North America and throughout Europe.
There were not sufficient articles from specific geographic regions such as Asia, Australia or South America to perform
informative statistical analyses on these individual areas. Moreover, we did not evaluate the “Other” category separately
as this group contains nations with significant ethnic/geographic diversity. Thus, the primary subgroup analyses were
limited to North America and Europe.
Additionally, all articles within a particular region were combined together into a single analysis regardless of the
detection method utilized (ie: PCR, ISH, p16, etc). The number of articles employing methods other than PCR was too
small to perform relevant statistical analyses assessing trends over time for each distinct detection method.
3
HPV prevalence was calculated for each article by dividing the number of patients with HPV-positive OPSCCs
by the total number of OPSCCs analyzed. Prevalence values were categorized into four time frames (pre-1995, 1995—
1999, 2000—2004 and 2005—present) on the basis of the median year of the study’s sample collection period. An
analysis of variance (ANOVA) model with time frame used as a four-level categorical variable and weighted by the total
number of OPSCCs analyzed in each paper was utilized to model the time trends of HPV in OPSCC. This analysis
included all articles for the worldwide analysis (articles=105, total OPSCCs=9541), with sub-analyses performed for
North America (articles=40, total OPSCCs=2844) and Europe (articles=47, total OPSCCs=4812). The p-values generated
from this analysis reflect the overall trend of HPV-positivity across the four time frames within each of these regions (pvalue for trend).
A more detailed evaluation was then performed to assess the differences in HPV prevalence between every
adjoining time frame from pre-1995 to 2005—present for North America and Europe. All analyses were performed using
the procedure PROC MIXED from SAS/STAT software (version 9.3). Plots were generated by the ggplot2 package in R
(version 3.0.2), and geographical maps were created with Google Fusion Tables (©2014 Google- Imagery ©2014 NASA,
TerraMetrics). Every statistical test was two-sided, and a p-value less than 0.05 was considered statistically significant.
Finally, we examined the present day prevalence of HPV in OPSCC within individual countries. Due to the
limited number of articles (less than three) for each country in the 2005—present time frame, we collated data from
studies with median year for sample collection between 2000—present. From all articles within one country, we
aggregated the total number of OPSCCs analyzed as well as the number of HPV-positive OPSCCs detected. The
proportion of HPV-positive OPSCCs and 95% confidence intervals were generated and plotted for each country using
Graphpad Prism v6.0d.
4
Supplemental Tables
Table S1. Final group of articles excluded from this meta-analysis.
Article
Reason for Exclusion
Country
Region
Shahnaz Begum et al, 200553
Failure to report sample collection period
United States
North America
United States
North America
United States
North America
United States
North America
United States
North America
United States
North America
United States
North America
United States
North America
Czech Republic
Europe
Denmark
Europe
Denmark
Europe
England
Europe
France
Europe
Germany
Europe
Germany
Europe
Odey C. Ukpo et al, 201151
RD Chernok et al, 200955
Justine M. Ritchie et al, 200356
Seth R. Schwartz et al, 200158
Anthony C. Nichols et al, 200959
Jose Jiron et al, 201461
Maura Gillison et al, 200827
R. Tachezy et al, 200563
Pernille Lassen et al, 200965
Pernille Lassen et al, 201367
AG Schache et al, 201352
Jean Lacau St Huily et al, 201169
Jens P. Klussmann et al, 200371
Jens P. Klussmann et al, 200373
Overlapping patient population with
James S. Lewis et al, 201054
Overlapping patient population with
James S. Lewis et al, 201054
Overlapping patient population with
Elaine M. Smith et al, 200457
Failure to provide number of HPVpositive OPSCCs detected
Overlapping patient population with
Anthony C. Nichols et al, 201060
Overlapping patient population with
Seema Sethi et al, 201262
Overlapping patient population with
D’Souza et al, 200728
Overlapping patient population with Jan
Klozar et al, 200864
Overlapping patient population with
Pernille Lassen et al, 201066
Overlapping patient population with
Pernille Lassen et al, 201066 and Pernille
Lassen et al, 201168
Overlapping patient population with AG
Schache et al, 201115
Overlapping patient population with Jean
Lacau St Guily et al, 201170
Overlapping patient population with
Niklas Reimers et al, 200772
Overlapping patient population with Jens
P. Klussmann et al, 200174
Claus Wittekindt et al, 200575
Failure to report sample collection period
Germany
Europe
Gerald Niedobitek et al, 199076
Failure to report sample collection period
Germany
Europe
Italy
Europe
Spain
Europe
Sweden
Europe
Sweden
Europe
Sweden
Europe
Sweden
Europe
Gianna Badaracco et al, 200077
Ignacio Alvarez Alvarez et al,
199779
Signe Friesland et al, 200180
Hanna Mellin et al, 200082
Hanna Mellin et al, 200383
Anders Nasman et al, 201284
Overlapping patient population with Aldo
Venuti et al, 200478
Overlapping patient population with JP
Rodrigo et al, 201417
Overlapping patient population with Lalle
Hammarstedt et al, 200681
Overlapping patient population with Lalle
Hammarstedt et al, 200681
Overlapping patient population with Lalle
Hammarstedt et al, 200681
Overlapping patient population with Lalle
Hammarstedt et al, 200681 and Anders
Nasman et al, 200918
5
Anders Nasman et al, 201385
Hanna Mellin Dahlstrand et al,
200586
Jos M.J.A.A. Streatmans et al,
200988
Peter J.F. Snijders et al, 199290
Harriet C. Hafkamp et al, 200392
Angela Hong et al, 201093
Wei Li et al, 200394
Angela Hong et al, 201096
Zeyi Deng et al, 201197
Kwonoh Park et al, 201399
Overlapping patient population with Lalle
Hammarstedt et al, 200681 and Anders
Nasman et al, 200918
Overlapping patient population with Lalle
Hammarstedt et al, 200681 and Hanna
Mellin et al, 200287
Overlapping patient population with
Harriet C. Hafkamp et al, 200889
Overlapping patient population with Peter
J.F. Snijders et al, 199491
Failure to report sample collection period
Overlapping patient population with
Angela Hong et al, 201011
Overlapping patient population with
Angela Hong et al, 201395
Overlapping patient population with
Angela Hong et al, 201011
Overlapping patient population with Zeyi
Deng et al, 201398
Overlapping patient population with Miji
Lee et al, 2013100
6
Sweden
Europe
Sweden
Europe
Netherlands
Europe
Netherlands
Europe
Netherlands
Europe
Australia
Other
Australia
Other
Australia
Other
Japan
Other
South Korea
Other
Table S2. Detailed information extracted from each article included in the meta-analysis.
North America
‡
Median
Year
# of HPV+ OPSCCs
28
102
103
104
57
105
106
107
1992
2003
1986, 1992,
1997, 2002
2002
2000
1985
1997
1995
2002
2000
1998
34
25
8/49 (1986), 27/75 (1992),
48/93 (1997), 33/46 (2002)
72
12
2
17
25
26
48
24
54b
Ref. #
21
101
12a
Total # of
OPSCCs
Material
Detection
Method
Country
60
30
Fresh frozen
FFPE
PCR
PCR
United States
United States
263
FFPE
PCR
United States
100
16
7
48
67
49
102
35
FFPE
FFPE
Fresh frozen
FFPE
FFPE
FFPE
FFPE
FFPE
United States
United States
United States
United States
United States
United States
United States
United States
2002
158
239
FFPE
c
108
109
110
2002
1999
1996
38
12
10
62
21
11
FFPE
FFPE
FFPE
111d
2001
27
42
FFPE
112
62
113
24
41
22
1/5 (1982), 3/7 (1987), 4/7
(1992), 15/20 (1997),
27/33 (2002)
9/24 (2002), 13/23 (2009)
52
81
55
FFPE
FFPE
FFPE
72
FFPE
PCR
United States
115
1991
2003
1992
1982, 1987,
1992, 1997,
2002
2002, 2009
ISH
PCR
Southern blot
ISH
PCR
ISH
PCR
PCR
p16 IHC, ISH,
PCR
ISH
ISH
PCR
PCR with flight
mass spectroscopy
PCR
PCR
PCR
47
ISH
United States
116
1989
14
22
PCR
United States
117
118
119
2003
1996
1988
206
15
2
323
29
14
ISH
PCR
PCR
United States
United States
United States
120
2001
13
14
FFPE
Fresh frozen,
FFPE
FFPE
Fresh frozen
Fresh frozen
Fresh frozen,
FFPE
PCR
United States
121
2006
37
50
FFPE
122
123
60e
124
1989
2004
2001
2009
48
23
53
111
79
31
68
162
125
2002
19
23
126
1994
12
34
f
2002
137
169
128
2001
16
22
129
2004
1996, 2002,
2008
2002
1997, 2006
2007
73
8/39 (1996), 43/63 (2002),
44/71 (2008)
12
1/7 (1997), 4/16 (2006)
11
111
FFPE
Fresh frozen
FFPE
FFPE
Fresh frozen,
FFPE
Information not
available
FFPE
Fresh frozen
and FFPE
FFPE
173
20
23
18
114
127
13
130
14
131
aDr.
PCR with flight
mass spectroscopy
PCR
PCR
ISH
p16 IHC
United States
United States
United States
United States
United States
United States
United States
United States
United States
United States
United States
United States
United States
ISH, PCR
United States
PCR
United States
PCR
Canada
PCR
Canada
PCR
Canada
FFPE
PCR
Canada
FFPE
FFPE
Fresh frozen
PCR
p16 IHC, PCR
PCR
Canada
Greenland
Mexico
Anil Chaturvedi sent us a table outlining the number of HPV-positive OPSCCs detected in each of the four time periods based on the Inno-LiPA
assay as this information was not provided for the 1990—1994 or 1995—1999 time periods.
7
bDr.
James Lewis provided the sample collection period for a different article.51 He then helped us identify studies with overlapping patient
populations from his group’s work to allow us to select the most representative study from his cohort of patients.54
cDr. Maura Gillison provided us with the sample collection period for this study.
dDr. Francis Worden provided us with the sample collection period for this study.
e
Drs. James Rocco and Anthony Nichols provided the sample collection period for a different article.59 Subsequently, they helped us select the most
inclusive analysis from studies of their patients to include in our analysis.60
fDrs. Bernard Fortin and Denis Soulieres provided us with the sample collection period for this study.
Europe
‡
Median
Year
# of HPV+ OPSCCs
Total # of
OPSCCs
Material
Detection
Method
64
2003
50
71
FFPE
PCR
132
2004
71
109
FFPE
PCR
133
2005
35
44
FFPE
PCR
68
66
83
53
5/36 (1992), 12/29 (2005),
17/30 (2008)
9
200
143
PCR
England
PCR
England
135a
1991
2
7
PCR
England
136
137
138
70
139
140
2003
1997
1998
2004
1994
1996
69
5
41
146
14
32
138
5
60
314
75
52
p16 IHC, PCR
PCR
p16 IHC
PCR
PCR
PCR
England
Finland
Finland
France
France
France
141
1996
24
138
PCR
France
b
1998
1
9
PCR
Germany
142
2006
12
24
PCR
Germany
143
144
72
145
2000
1998
1999
1998
24
7
30
12
31
16
106
24
PCR
PCR
PCR
PCR
Germany
Germany
Germany
Germany
146
2004
13
52
FFPE
FFPE
Fresh frozen
and FFPE
FFPE
Fresh frozen
and FFPE
FFPE
Fresh frozen
FFPE
FFPE
FFPE
FFPE
Fresh frozen
and FFPE
Fresh frozen
Fresh frozen
and FFPE
FFPE
Fresh frozen
FFPE
Fresh frozen
Fresh frozen
and FFPE
p16 IHC
p16 IHC
134
1995
1988
1992, 2005,
2008
2005
Czech
Republic
Czech
Republic
Czech
Republic
Denmark
Denmark
p16 IHC, PCR
Germany
147
1996
51
162
PCR
Germany,
Netherlands
78c
1998
6
14
148
149
150
151
16
17
18
152
2009
2006
1994
1995
1972, 1990
1994, 2004
2004, 2006
2004
153
1999, 2002,
2004, 2006
10
66
17
9
24/63 (1972), 47/74 (1990)
3/166 (1994), 5/82 (2004)
40/52 (2004), 43/46 (2006)
12
15/26 (1999), 18/24
(2002), 22/26 (2004),
16/19 (2006)
7/30 (1974), 12/42 (1984),
48/84 (1994), 32/47 (2001)
60
15
Ref. #
15
74
81
154
155
1974, 1984,
1994, 2001
2003
1997
95
14
FFPE
Country
PCR
Italy
21
120
90
78
137
248
98
69
Fresh frozen
and FFPE
Fresh frozen
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
PCR
p16 IHC
PCR
PCR
PCR
p16 IHC, PCR
PCR
PCR
Italy
Italy
Italy
Italy
Norway
Spain
Sweden
Sweden
95
FFPE
PCR
Sweden
203
FFPE
PCR
Sweden
77
25
FFPE
Fresh frozen
PCR
PCR
Sweden
Sweden
8
156
157
87
158
159
160
89
91d
161e
162
10
2002
2003
1994
1986
1994
1995
1996
1987
1997
2003
1990, 1995,
2000, 2005,
2009, 2010
19
153
12
10
14
35
33
12
7
150
2/39 (1990), 3/37 (1995),
6/42 (2000), 12/59 (2005),
24/86 (2009), 18/62 (2010)
45
211
22
25
99
85
81
14
30
711
Fresh frozen
FFPE
Fresh frozen
FFPE
FFPE
FFPE
FFPE
FFPE
Fresh frozen
FFPE
PCR
p16 IHC, PCR
PCR
PCR
PCR
p16 IHC
ISH
PCR
PCR
p16 IHC, PCR
Sweden
Sweden
Sweden
Sweden
Switzerland
Switzerland
Netherlands
Netherlands
Netherlands
Netherlands
325
Fresh frozen
and FFPE
p16 IHC, PCR
Netherlands
Total # of
OPSCCs
Material
Detection
Method
Country
280
FFPE
p16 IHC, PCR
Australia
403
p16 IHC, PCR
Australia
PCR
Brazil
PCR
PCR
ISH
PCR
PCR
PCR
PCR
ISH
ISH
PCR
PCR
PCR
PCR
p16 IHC, ISH,
PCR
PCR
Brazil
India
India
Israel
Japan
Japan
Japan
South Korea
South Korea
South Korea
Taiwan
Taiwan
International
aDr.
John Field provided us with the sample collection period for this study.
Jens Klussmann provided us with the sample collection period for this study.
cDr. Aldo Venuti provided us with the sample collection period for this study.
dDr. Peter Snijders provided us with the sample collection period for this study.
eDr. Ruud Brakenhoff provided us with the sample collection period for this study.
bDr.
Other
Ref. #
‡
Median
Year
# of HPV+ OPSCCs
11a
1988, 1993,
1998, 2003
95
1996
6/31 (1988), 11/52 (1993),
44/111 (1998), 40/86
(2003)
185
163
2003
6
91
164
165
166b
167
98
168
169c
100
170
171
172
173
174
1995
2010
1999
1984
2008
2003
1986
2005
2002
1998
1998
2001
2001
4
24
13
4
24
23
7
59
20
10
14
69
56
11
105
42
13
48
71
13
89
56
38
111
92
111
FFPE
Fresh frozen
and FFPE
Fresh frozen
Fresh frozen
FFPE
FFPE
Fresh frozen
FFPE
Fresh frozen
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
175
2003
88
169
FFPE
176
1997
26
142
Fresh frozen
aDrs.
International
International
Barbara Rose and Angela Hong provided us with the number of OPSCCs analyzed in each of their discrete time periods since their study only
described the percent that were HPV-positive without providing the total number of cancers evaluated.
bDr. Rekha Kumar provided us with the sample collection period for this study.
cDrs. Hiroyuki Mineta and Kiyoshi Misawa provided us with the sample collection period for this study.
‡
Median year: Median year of each study’s sample collection period.
9
Table S3. Number of articles and OPSCCs analyzed within each country.
Country
United States
Canada
Greenland
Mexico
Czech Republic
Denmark
England
Finland
France
Germany
Germany,
Netherlands
Italy
Norway
Spain
Sweden
Switzerland
Netherlands
Australia
Brazil
India
Israel
Japan
South Korea
Taiwan
International
Articles
(n)
Total
OPSCCs (n)
33
5
1
1
3
2
4
2
4
7
2308
495
23
18
224
343
254
65
579
262
1
162
5
1
1
10
2
5
2
2
2
1
3
3
2
3
323
137
248
870
184
1161
683
102
147
13
132
183
203
422
10
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