Human Papillomavirus infection is not a major risk
factor for ESCC and GCA in high risk area in China.
Wenqiang Wei et.al
Dept. of Cancer Epidemiology, Cancer Institute / Hospital CAMS
Genetic Epidemiology Branch, DCEG, National Cancer Institute
Dept. of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University
Dept. of Applied Tumor Biology, Institute of Pathology, University of Heidelberg
Outline
1.Background
2.Studies from Our Group
Background
1. Esophageal squamous cell carcinoma (ESCC) is the
fourth leading cause of cancer death in China.
2. HPV infection has been suggested to have a role in
the etiology of ESCC. HPV vaccine given the
potential for prevention through use of the vaccine if
HPV is involved.
3. But…., the results of previous studies have not been
consistent.
研究地区
检测方法
HPV 型别
HPV(+)%
样本量
作者
PCR
通用引物,16,18
78
265
Cao 2005
PCR
通用引物
59.1
110
何保昌 2005
Microarray
19种
11.7
94
Dai 2007
ISH
6,11,16,18,30,53
16.9
700
Chang 2000
PCR
6,11,16,18
49
51
Chang 1990
PCR
6,11,16,18,33
0
35
陆士新 1995
磁县
PCR
通用引物
20.3
128
刘艳丽 2003
汕头
PCR
6,11,16,18
65.5
176
Shen 2002
PCR
-
64.5
318
陈玲 2008
INNO-LiPA
24种
21
67
Lu 2008
南非
PCR
通用引物
46
50
Matsha 2002
肯尼亚
PCR
27种
0
29
White 2005
伊朗
PCR
16,18,31,33…
23.6
140
Far 2007
印度
CP,16,18
26.7
101
Katiya 2005
通用引物
15.8
165
Damin 2006
13种高危型
2.5
40
Weston 2003
智利、哥伦
比亚
墨西哥
PCR
auto-nested
PCR
hc2
PCR,
Southern blot
PCR
通用引物
73
29
Castillo 2006
6,11
88
17
Acevedo 2004
匈牙利
PCR
6,11,16,18,52,58,66,73
82
39
Szentirmay 2002
意大利
PCR
通用引物,16,18
0
45
Talamini 2000
瑞典
PCR
16, 18, 31, 33…
16
100
Dreilich 2006
韩国
PCR
16,18,31,33…
0
102
Koh 2007
北京
ISH
16,18,31,33…
0
83
Suzuk 1996
安阳
林县
高
发
区
新疆
巴西
低
发
区
Background
The argument support for HPV involvement in esophageal
carcinogenesis:
• biologic similarities between the esophagus and the
cervix (a squamous mucosal surface)
• shared exposure of esophageal and oral epithelium, as
an association between HPV and tonsillar cancer has
already been established
• HPV detection in esophageal cancer tissues and
precursor lesions
• In vitro evidence of HPV-induced transformation of
esophageal cells.
Background
The argument against the involvement of in esophageal
carcinogenesis:
• high variability in HPV-positivity among esophageal
cancer cases(0%-100%)
• Some have argued that any true association between
HPV and esophageal cancer would not vary
geographically since HPV is a common infection
worldwide
• inconsistent serologic risk estimates, with estimates
from the largest studies tending toward the null
• lack of documented associations between ESCC and
sexual behavior, immunosuppression, or previous HPVassociated malignancy.
2. Studies from Our Group
• Cross sectional study
• Prospective study
• Multi-center Lab Study
Cross sectional study
• Objective: HPV infection distribution in High risk area of
ESCC in Linxian, China.
• Balloon cytology
• HC-II methods (13 HR,5 LR types)
Cross sectional study
Conclusion from Cross sectional study
• High-risk HPV types in 12% of subjects with no evidence
of squamous dysplasia and a similar proportion of
individuals with mild, moderate and severe dysplasia.
• Suggest that HPV infection is not a major risk factor for
ESCC in this high-risk population.
No association between HPV infection and the neoplastic progression of esophageal squamous cell
carcinoma: Result from a cross-sectional study in a high-risk region of China.
International Journal of Cancer: 119: 1354–1359, 2006
Prospective study
•
Baseline serum sample, ELISA assays for detecting antibodies
•
prospectively examined potential associations between HPV 16, HPV
18 and HPV 73 and the occurrence of upper gastrointestinal cancers
•
Case and control subjects were selected from the 29,584 participants
of the Linxian General Population Trial.
•
Prediagnostic serum samples from 99 cases of ESCC, 100 cases of
GCA, 70 cases of GNCA, and 381 age- and sex- matched controls
were selected for this study.
Prospective study
• Fewer than 15% of ESCC, GCA, or GNCA cases were
positive for each HPV type, and no significant
associations were found.
• Do not support a major role for these HPV types in the
etiology of esophageal and gastric cancers in Linxian.
International Journal of Cancer: 119: 579-584, 2006
Summary of the potential reason from
previous study
The variability may comes from:
• Small study sizes
• inter-laboratory variability
• differences in laboratory detection methods
• contamination during sample collection or
laboratory processing
Multi-center lab study
Objective
The overall objective is to test the hypothesis that HPV is
involved in the pathogenesis of ESCC/GCA in Linxian, China.
1.
To determine the prevalence of HPV DNA in ESCC/GCA
2.
To determine the activity of HPV in HPV DNA positive
cases
Methods
1. Subjects recruitment
• ESCC /GCA cases finished the informed consent, Demographic
data were abstracted from medical records.
• Blood sample and resection specimen collection followed by
rigorous sterile procedures
2. Specimen Collection and Processing
• Careful sterile technique, including sterile equipment, gloves,
and sleeve covers, was used to prevent contamination while
processing specimens from ESCC/GCA cases.
Methods
3. HPV DNA Testing
• Frozen tissue - the Roche HPV Linear Array Assay.
• Formalin-fixed paraffin embedded tissue specimens
- SPF10 LiPA25 version 1
4. Evaluation of HPV Oncogene Activity
• Cases positive for HPV DNA - immunohistochemical
analysis of p16INK4a over-expression.
Result - ESCC
Table 1. Distribution of characteristics among 272 esophageal
squamous cell carcinoma cases in Linxian, China
N
%
Currently drinking alcohol, yes
27
9.9
Ever drank alcohol, yes
110
40.4
Currently smoking, yes
22
8.1
Ever smoked, yes
160
58.8
Gender, Male
187
68.8
Marital status, Married
266
97.8
Family history of cancer, yes
67
24.6
Patients came from 13 of the 31 Provinces of
China including both the high-risk Taihang
mountain region (27.6%) and other areas
(72.4%).
Result - ESCC
Table2 The prevalence of HPV infection on ESCC
HPV-DNA
+
Frozen Tissue
Formalin-fixed
paraffin-embedded
tissue
1 (0.4%)
2 (0.7%)
266
(99.6%)
265
(99.3%)
Total
267@
267
@ The β-globin signal was absent for 5 cases (1.8%).
267cases have the adequacy of the DNA
# One male case HPV89 weakly positive.
$ One female for HPV16 and one male for HPV31.
Result - ESCC
 None of the three HPV DNA-positive cases
exhibited p16INK4a protein over-expression.
 The HPV16-positive case was sero-negative for
HPV16 E6 and E7 antibodies.
* No role for human papillomavirus in esophageal squamous cell carcinoma in
China.Int J Cancer. 2010 Jul 1;127(1):93-100
Result - GCA
Table 2. Distribution of characteristics among 144 GCA in
Linxian, China
Result - GCA
 The β-globin signal was strong for 71% of cases (n =102), weak
for 4% (n = 6), and absent for 25% (n = 36).Thus, β-globin, and
therefore DNA quality, was adequate in 75% (108 of 144) of
cases.
 Among the 108 cases with adequate β-globin, all were negative
for HPV DNA by Linear Array and E6/E7-based PCR (100%; 95%
CI 97-100%).
*The gastric cardia is not a target for human papillomavirus-induced carcinogenesis. Cancer Epidemiol
Biomarkers Prev. 2010 Apr;19(4):1137-9. Epub 2010 Mar 23.
Conclusion
1. This study provides the most definitive evidence that HPV is not
involved in ESCC carcinogenesis
2. HPV DNA contamination cannot be ruled out as an explanation for
high HPV prevalence in ESCC tissue studies with less stringent
tissue collection and processing protocols.
3. HPV infection does not contribute to gastric cardia carcinogenesis
in this part of China.
Acknowledgement
NCI NIH
CI/H CAMS
Dr. Christian C. Abnet
Dr. Sanford M. Dawsey
Dr. Philip R. Taylor
Dr. Farin Kamangar
Dr. Jill Koshiol
Dr. Mark J. Roth
Dr. Philip E. Castle,
Dr. Youlin Qiao
Dr. Wen Chen
Dr. Jian-Song Ren
Dr. Jian-Bing Wang
Dr. Zhi-Wei Dong
University of Heidelberg
Dr. Magnus von Knebel Doeberitz
Svetlana Vinokourova, PhD
The Johns Hopkins University
Dr. Patti Gravitt, PhD
Dr. Rapheal Viscidi, MD
Thanks for you time
高发区林县拉网细胞中HPV感染情况
HPV感染率（%）
检测方法
HPV型别
正常
不典型增生
癌
样本数
作者
chang 1990
FISH
6,11,16,18
9(22)
65(72)
6(67)
80
ISH
16
72(21)
63(78)
3(100)
138
Li 2001
PCR
16
72(60)
63(84)
3(100)
138
HC2
13种
475(13)
223(9)
4(0)
702
Gao 2006
PCR
14种
57(7)
-
-
57
Peixofo 2001
PCR
16,18
357(57)
-
-
357
Cao 2005
Linear Array Kit
Using PGMY primer, detect 37 HPV types.
• High risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68,
73, 82, 26, 53, 66
• Low risk: 6, 11, 40, 42, 54, 55, 61, 62, 64, 67, 69, 70, 71, 72,
81, 83, 84, IS39, CP6108
样品准备：病理组织切片（三明治法）
阴性对照
3x 4 μm
HE
食管癌病理组织 1x 4 μm
•
HPV PCR HPV PCR BU
3x8 μm
3x 8 μm
p16
1x 4 μm
HE
1x 4 μm
An HPV negative quality control block was cut
between every 10 ESCC blocks
SPF10 PCR 系统
•
•
14种高危型别：16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
11种低危型别： 6, 11, 34, 40, 42, 43, 44, 53, 54, 70, 74
•
•
通用引物，扩增50多种HPV型别
蜡块提取DNA，容易断裂。PCR片段(65bp)不易受DNA降解的影响
Kleter B. et al. Journal of Clinical Microbiology, 1999; 37(8):2508-2517
LiPA HPV分型检测
• LiPA HPV分型检测技术是以逆杂交为基础。
• 使用SPF10引物扩增HPV基因组L1区，随后合成的生物素
酰化的扩增子变性，与特异的寡聚核苷酸探针杂交。
• 杂交和冲洗后，加入抗生物素蛋白链菌素结合的碱性磷酸
酶，与先前的杂交物结合。与BCIP/NBT色原体孵育后产
生紫色沉淀物。可以检测 25种 HPV型别。
LiPA HPV Genotyping
11 16 54 35 42 51 68 70
单个型别感染
6
6 16 11 35 53 52 18
11 51 66 43 66 70 56
多重感染
高危 HPV 型别: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
低危 HPV 型别: 6, 11, 34, 40, 42, 43, 44, 53, 54, 70, 74
免疫组化检测 p16INK4a 过表达
• Dako: CIN tec™ p16INK4a Histology Kit
• 石蜡包埋活检组织：4μm
• 质量控制:
★ 使用CICMAS的肿瘤组织作为阳性对照，正常组
织为阴性对照。
★ 其中一个样品组织切片加阴性对照试剂。