Clinical Trial Details
— Status: Enrolling by invitation
Administrative data
| NCT number |
NCT01079546 |
| Other study ID # |
TASMC ZG09 156 CTIL |
| Secondary ID |
|
| Status |
Enrolling by invitation |
| Phase |
N/A
|
| First received |
March 2, 2010 |
| Last updated |
March 2, 2010 |
| Start date |
January 2010 |
| Est. completion date |
January 2011 |
Study information
| Verified date |
March 2010 |
| Source |
Tel-Aviv Sourasky Medical Center |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
Israel: The Israel National Institute for Health Policy Research and Health Services Research |
| Study type |
Observational
|
Clinical Trial Summary
The rationale for this study proposal is derived from previous case series demonstrating
that up to 60% of patients with HNSCC, especially in the oropharynx are associated with high
risk HPV infection.
In this study, we will characterize the association between HPV infection and HNSCC in
Israel and establish a program for its diagnosis and management based on HPV as a biomarker.
The rationale for the proposed research is that once it is known which types of HPV are
associated with HNSCC, this information can be used to direct diagnosis and screening effort
to high risk population. Our approach is based on establishing a multicenter consortium of
leading researchers that will establish a joint database of demographic, clinical and
biological data from various regions in Israel. For this we have assembled a
multidisciplinary research team with the scope and breath (surgical oncology, pathology,
virology and molecular biology) needed to complete all phases of the research successfully.
The research will be coordinated and performed at the Applied Cancer Research Laboratory,
Tel Aviv Sourasky Medical Center. The patients will be recruited from 7 tertiary medical
centers in Israel: Ichilov, Bellinson, Hadassa, Rambam, Soroka, Sheba and Nazeret..
Description:
To determine the prevalence and classify the high risk human papillomavirus types associated
with head and neck cancer in Israel. We will determine the HPV genotype distribution in
Israel in order to study the ethnic uniqueness of the region. Many studies have shown HPV 16
to be the most prevalent type in HNSCC (NEJM). In Israeli Jewish women the HPV types 16, 39,
52, and 18 were the most prevalent genotypes found (15). In our aim, we will determine the
prevalence of various HPV genotypes in HNSCC specimens.Study population The group will
include patients treated with primary surgery or biopsy, with or without adjuvant
radiotherapy between 1998 and 2010 for SCC of the oral cavity, oropharynx, hypopharynx,
larynx and paranasal sinuses.
The anatomic site and extent of the primary tumor will be documented. The TNM classification
of will be based on the staging system revised by the American Joint Committee on Cancer
(AJCC). Patients will be recruited from seven national cancer referral hospitals, including
Soroka Medical Center in the Negev (Beer Sheva), Sourasky Medical Center (Tel Aviv), Rabin
Medical Center (Petach Tikva), Hadassa Medical (Jerusalem), Rambam Medical Center (Haifa),
Sheba medical center (Ramat- Gan) and French Hospital Nazareth (Nazareth). These centers
cover almost the entire population in Israel, including Israeli Jews, Bedouins, Druze and
Israeli Arab population.
Data Entry Data will be entered into a commercially available spreadsheet (Microsoft Excel
2000, Microsoft Corporation, Seattle, WA) and statistical analysis will perform using a
computerized software package (SPSS). We have previously used a similar system to report the
results of 146 patients with skull base sarcomas in an international multicenter study
including 17 cancer centers (17).
Tumor specimens Specimens will be collected retrospectively, and comprise of paraffin
sections of primary tumors or biopsies retrived from the pathology department archives at
each center. Transportation of paraffin blocks to Tel Aviv for deparaphinization and HPV
testing will take place personally by the investigators. Histopathological analysis Primary
tumor specimens will be first evaluated by pathologists at each participating cancer. All
specimens will be than re-analyzed and evaluated by head and neck pathologist, Dr. Kaplan
Ilana. Specimen dissection and tissue sampling of the primary tumor will be in accordance
with the current guidelines for the histopathological assessment of head and neck cancer
carcinoma.
HPV detection at the laboratory Tissue sections will be subjected to deparaffinization,
heatinduced target retrieval, and digestion with proteinase K (Roche Diagnostics,
Indianapolis, IN), as described previously (18). DNA is then purified from paraffin by
deparaffinization, proteinase K digestion, phenol/chloroform extraction, and ethanol
precipitation. We will use the HPV genotyping system using a PCR based HPV GenoArray test
kit for genotyping of 37 HPV taypes (Hybribio Limited, Hong Kong). Specimens are placed into
PreservCytR LBC medium (ThinPrep liquid Pap vial; Cytyc Corporation). The Amplicor HPV test
kit contains a pool of HPV-specific primers designed to amplify HPV DNA from 13 HR genotypes
(types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). According to the
manufacturer's specification, the Amplicor HPV test detects HPV genotypes 31, 52, 58, and 59
at 240 copies/ml and HPV genotypes 16, 18, 33, 35, 39, 45, 51, 56, and 68 at 100 copies/ml
with a positivity rate greater than 95%. All genotypes are detected with a 100% positivity
rate at 480 copies/ml.
HPV genotyping HPV genotyping is performed by using a commercially available PCR based HPV
GenoArray test kit (Hybribio Limited, Hong Kong), which makes use of both DNA amplification
and HybriBio's proprietary flowthrough hybridization technique to simultaneously identify 21
HPV genotypes, including 5 low-risk types (types 6, 11, 42, 43, and 44), 14 HR types (types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), and 2 intermediate-risk types
(types CP8304 and 53).
The test employs a macroarray format with a nylon membrane onto which HPV genotype-specific
oligonucleotides probes have been immobilized. In Situ Hybridization system will be
performed with Amplicor HPV test (Roche Molecular Systems) for validation of the results.
This kit is designed for the detection and discrimination of human papilloma virus (HPV) DNA
in paraffinembedded tissue. An HPV16-positive tumor specimen will be used as a positive
control.
p16 Immunohistochemistry
The expression status of p16 is strongly correlated with tumor HPV status (19) and therefore
we will evaluate the presence of this protein by immunohistochemistry (20). After 5- μ m
sections are deparaffinized, antigen retrieval is performed by use of heat-induced epitope
retrieval with 10 mM citrate buffer. Immunostaining will be performed with a Ventana
automated stainer (Ventana Medical Systems, Tucson, AZ) according to the manufacturer's
recommendations. Cervical specimen known to be positive for HPV will be used as positive
control and normal tissues as baseline controls. A mouse monoclonal antibody against p16
(MTM Laboratories, Heidelberg, Germany) at 1:500 dilution will be utilized. The p16 antibody
is detected using the avidin-biotin-peroxidase technique (Dako LSAB Kit, Dako). On
histopathologic review, the pattern of p16 expression is generally dichotomous according to
tumor sample, with p16 staining either absent (negative) or present with strong and diffuse
nuclear and cytoplasmic staining (positive). Relative protein expression will be estimated
using dedicated software (Metamorph, Molecular Devices, Union City, CA), which analyzed the
intensity and area of positive staining. Immunohistochemical interpretation will be
performed by two investigators blinded to the HPV status and identity of the patient from
whom the tumor originated.
Evaluation the association between HPV status and therapeutic response or survival in
patients with oropharyngeal/laryngeal SCC Prospective analyses of case series in the United
States and Europe have consistently demonstrated that patients with oropharyngeal SCC which
are HPV-positive have a better prognosis than patients whose tumors are HPV negative
(21,22). We will evaluate the effect of tumor HPV status on treatment response and survival
outcomes among patients with oropharyngeal and laryngeal SCC who were treated with primary
chemoradiation or radiation therapy (organ preservation regime).
Data Collection Each center will collect data from patients charts. The data will include:
age, sex, ethnic group, area of birth and residence, religion, education (ever attendance at
school, number of years of school attendance, and age at leaving school), longest
occupation, use of tobacco in its different forms, alcohol drinking habits, dietary habits,
marital status, sexual history (number of lifetime sexual partners, visits to prostitutes,
and frequency of oral sex), histories of various diseases, family history of cancer, and
oral cavity health. A "smoker" is defined as an individual who reports having smoked tobacco
daily for at least 1 year, and smokers will be asked about duration of smoking and amount
and type of tobacco smoked (cigarettes, cigars, pipes or any form of tobacco chewing). A
"drinker" is defined as an individual who reports drinking alcoholic beverages at least once
a month. Details will be obtained on types of beverages, amounts and duration of habitual
drinking. information on tumor-node-metastasis classification and staging will be obtained
from the study patients. This classification will be converted into stage according to the
American Joint Committee on Cancer (AJCC) Cancer Staging Manual. We will evaluate the
outcome of each therapy in terms of response (complete response, partial response and
progressive disease according to the RECIST classification)
Statistical analysis Five-year overall survival (OS), disease-specific survive al (DSS) and
locoregional control rates will be calculated using the Kaplan-Meier method and the
difference in survival rate will be assessed by a log rank test (17). OS will be measured
from the date of surgery to the date of death or last follow-up. For DSS, the patients who
died from causes other than SCC will be censored at the time of death. The variables that
will have prognostic potential suggested by univariate analysis will be subjected to
multivariate analysis with the Cox proportional hazards regression model. All statistics
will be two-sided. A value of P< 0.05 will be considered to indicate statistical
significance. Comparison of HPV/p16 classification (three-class model) will be performed by
Kruskal-Wallis analysis followed by Wilcoxon rank sum test with Bonferroni corrections.
Comparison of protein expression by HPV/p16 classification will be made using one-way
analysis of variance (ANOVA) with post hoc comparisons by Dunnett's T3 test. Comparison of
HPV - three class model classification status with specific clinical and pathologic
variables (sex, TNM stage, T stage, N stage, histologic grade, management, radiotherapy,
chemoradiation, alcohol use, tobacco use and clinical response), will be performed by
Fisher's exact test. Clinical response will be recorded as complete response versus no
response/partial response after completion of treatment. All calculations and analyses will
be performed with the Statistical Package for the Social Sciences version 11.5 for Windows
(SPSS Inc, Chicago, IL), by a qualified statistician at Tel Aviv Souraky Medical Center
(Esther Shabtai PhD).
Sample size and its justification The prevalence of HNSCC in Israel is estimated to be 52
oropharyngeal and 250 laryngeal cancer new cases per year (Middle East cancer consorcium,
www.mecc.cancer.gov). The medical centers that will participate in this study cover 80% of
the population of the State of Israel. we estimate that the participation rate among case
patients will be 89% (range from 76 to 99.0%) (REF JNCI International). Therefore it is
estimated a study period of 6 months approximately 100 samples will be recruited.
