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Human papilloma virus-16 (HPV-16) Infection Association with CIAP-2
Expression in Head and Neck Cancer.
Article    in  Medic al Onc olog y · Januar y 2011
DOI: 10.1007/s12032-013-0766-6
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Medical Oncology

ISSN 1357-0560

Med Oncol
DOI 10.1007/s12032-011-0143-2Human papillomavirus-16 (HPV-16)
infection association with CIAP-2
expression in head and neck cancer
Amal Mansour, Marwa Ali, Hussein
Helmy & Samar Kassim

123
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ORIGINAL PAPER
Human papillomavirus-16 (HPV-16) infection association
with CIAP-2 expression in head and neck cancer
Amal Mansour •Marwa Ali •Hussein Helmy •
Samar Kassim
Received: 24 November 2011 / Accepted: 15 December 2011
/C211Springer Science+Business Media, LLC 2012
Abstract Human papillomavirus (HPV-16) E6 proteins
inhibit apoptosis in both p53-dependent and p53-indepen-
dent manners. So it was relevant to assess the impact of
such infection on head and neck cancers and its relation to
the inhibitors of apoptosis (IAPs). CIAP2 is one of these
IAPs that is postulated to upregulated by E6 proteins of
HPV-16 by amplification of the locus bearing it in many
tissues. In this study, we aimed to search for the amplifi-
cation of the locus bearing CIAP-2 and its relation to HPV-
16 in head and neck cancer that may have prognostic and
therapeutic impacts on these patients. Total 30 patients
diagnosed as head and neck cancer (2 tissue samples were
taken from each patient: from the tumor and from the
safety margin). All samples were subjected to qualitative
polymerase chain reaction analysis for HPV-16 and qual-
itative and semiquantitative reverse transcription poly-
merase chain reaction of CIAP-2. There was a significant
association between HPV-16 and CIAP2 positivity and
head and neck tumors ( P=0.01). CIAP-2 expression in
malignant tissues was highly associated with HPV-16
infection with 73.9% sensitivity and absolute specificity.
Keywords Human papillomavirus /C1IAPs /C1CIAP /C1
Apoptosis /C1RT-PCR /C1PCRIntroduction
Head and neck squamous cell carcinoma (HNSCC) make
up nearly 90% of head and neck cancers [ 1]. In Egypt, the
annual death rates for malignant neoplasm of the pharynx
were 279 per 1,00,000 person/year for males and 283 per
1,00,000 person/years for females as reported in world
health statistics annual, 2010 [ 2]. Human papillomavirus
(HPV) was found to be highly associated with several types
of SCC at different sites which are: cervix, esophagus,
conjunctiva, and paranasal sinuses [ 3,4]. Studies reporting
treatment outcomes for head and neck cancer must now
account for HPV-16 status, because of its association with
improved prognosis [ 5].
New data from case–control studies showed an overall
prevalence of HPV-16 infection of 25.9% in specimens
obtained from patients with head and neck squamous cell
carcinoma that had been analyzed in 60 separate studies
[6]. However, there are many unresolved questions con-
cerning the pathogenesis of HPV-16 in head and neck
carcinoma [ 7]. It is possible that HPV-16-associated head
and neck squamous cell carcinoma arises by a different
mechanism from that involved in the pathogenesis of
HPV-16-associated cervical carcinoma [ 8].
Many studies were indicating a positive association
between HPV-16 and high-grade cancer lesions and sug-
gest that additional cofactors, such as cigarette smoking
and alcohol abuse, may be required as carcinogens to
advance HPV-16-infected cells toward neoplastic pro-
gression [ 7,9].
In the last decade, basic cancer research has produced
remarkable advances in our understanding of cancer biol-
ogy and cancer genetics. Among the most important of
these advances is the realization that apoptosis and the
genes that control it have a profound effect on the
A. Mansour ( &)/C1M. Ali /C1S. Kassim
Medical Biochemistry Department, Faculty of Medicine,
Ain Shams University, Abbassia, Cairo 11381, Egypt
e-mail: amal.bary@gmail.com
H. Helmy
Otolaryngology Department, Faculty of Medicine,
Ain Shams University, Cairo, Egypt
123Med Oncol
DOI 10.1007/s12032-011-0143-2
Author's personal copy

malignant phenotypes. As well as, deregulated apoptosis is
a hallmark of cancer [ 10,11]. Mammalian ‘‘inhibitor of
apoptosis’’ (IAP) proteins, such as CIAP-2 (cellular
inhibitor of apoptosis-2) CIAP-1 and XIAP (X linkedinhibitor of apoptosis), are key negative regulators of
apoptosis that can prevent apoptosis by directly inhibiting
caspases or by blocking pathways that activate them [ 12].
Amplification of the locus bearing CIAP-1 and CIAP-2
has been observed in liver, lung, and cervical tumors in
humans, raising the possibility that inhibition of cell death
by IAP proteins contributes to oncogenesis [ 13].
In this study, we aimed to search for the amplification of
the locus bearing CIAP-2 and its relation to HPV-16 in
head and neck cancer that may have prognostic and ther-
apeutic impacts on these patients.
Patients and methods
Patient’s population
Totally 60 tissue samples were obtained from 30 consecutive
patients that were recruited from the ENT Department, Ain
Shams University hospitals, Cairo, Egypt (from September2008 to February 2009).They were diagnosed as head and
neck cancer by the routine histopathology examination in the
Histopathology Department-Ain Shams University. Theywere enrolled in the present study after obtaining informed
consent. The enrolled patients are clinically staged according
to TNM classification [ 14]. Their mean age ±SD was
52.9 ±14.2 years, and the range was from 14 to 72 years.
All patients were sex matched (15 males and 15 females) and
20 of them were smokers (66.6%). Eighteen samples (60%)were diagnosed by histopathology as squamous cell carci-
noma (SCC) and 12 (40%) as invasive SCC. Twenty-four
(80%) were low grade by histopathological grading [ 15]. As
regards their TNM staging, 19 (63.3%) were early stage
tumors, while the rest 11 (36.7%) were late stage tumors.
Collection of samples
Sixty tissue samples from 30 patients diagnosed as head
and neck cancer were taken under general anesthesia for
PCR and RT-PCR analysis. They were classified into 30
malignant tissues (group 1) and 30 safety margin tissues ofthe same patients (group 2).
DNA and RNA extractionAll samples were obtained on a Petri dish and were chilled
on ice and then cut into small pieces (0.3 gm) for totalRNA and DNA extraction according to TriFast
TMPurifi-
cation Kits, based on a modified salt precipitationprocedure in combination with highly effective inhibitors
of RNase activity. (PeQLab Biotechnologie GmbH Cor-
poration, Erlangen, Germany). Extracted DNA and RNA
were used for the detection of HPV-16 DNA using quali-tative PCR and CIAP-2 RNA using qualitative and semi-
quantitative RT-PCR.
HPV-16 detection by PCR
The amplification of the gene coding for HPV-16 required
2lg of purified DNA and 25 pmol/ ll of each primer, and
their sequences were as follow: sense primer 5
0-CAAAGC
CGTCGCCTTGGGCA-30, and antisense primer 50-GGTGT
GGCAGGGGTTTCCGG-30(Access number NC_001526.1)
[16]. First step of PCR activation was done at 95 /C176C
for 10 min, repeated 35 cycles of denaturation at 94 /C176C for
1 min, annealing at 58 /C176C for 1 min, extension at 72 /C176C for
1 min, and then final extension at 72 /C176C for 10 min. The
amplified HPV-16 DNA products were found at 88 bp. That
was separated on 2% agarose gel and visualized by ethidium
bromide staining. In all the runs, 1 ll of DEPC-treated water
was used instead of the DNA sample as negative control
(Fig. 1).
Semiquantitative multiplex RT-PCR for CIAP-2 gene
It is a demanding amplification technique, which allows the
simultaneous detection of several RNA targets in a single
Fig. 1 a PCR analysis for HPV-16 positivity against HPV-16
negative cases as control group, DNA product at 88 base pairs (bp)
by agarose gel electrophoresis and ethidium bromide staining: lane 1
molecular weight ladder standard (50–1,000 bp), lane 2 PCR negative
control, lanes 3–8 positive HPV-16 samples, and lane 9 negative
HPV-16 sample. bRT-PCR analysis for CIAP-2 product at 385 bp
andb-actin at 300 bp by agarose gel electrophoresis and ethidium
bromide staining: lane 1 molecular weight ladder standard
(50–1,000 bp), lane 2 safety margin sample showing negative
CIAP-2 and positive bactin, lane 4 PCR negative control, and lanes
3, 5, 6, 7 CIAP-2 positive samplesMed Oncol
123
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tube. CIAP -2and housekeeping bactin primers were
designed, and multiplex RT-PCR was done for them; 2 lg
of RNA was converted to cDNA using QIAGEN One Step
RT-PCR Kit (QIAGEN, USA) by using CIAP-2 primers25 pm/ ll of each of the following primers: sense primer;
5
0-ACTTGAACAGCTGCTATCCACATC- 30and the
antisense primer; 50-GTTGCTAGGATTTTTCTCTGAA
CTGTC-30[17]. 3 pm/ ll were used from each of the b
actin primers (the sequences of these primers were chosen
according to Smith et al. [ 18]. Its sense primer was 50-CT
ACGTCGCCCTGGACTTCGAGC-30and antisense primer
was 50-GATGGAGCCGCCGATCCACACGG-30. First
step of RT was done at 60 /C176C for 60 min, PCR activation
was at 95 /C176C for 15 min, repeated 40 cycles of denaturation
at 95/C176C for 1 min, annealing at 50 /C176C for 1 min, extension
at 72/C176C for 1 min, and then final extension at 72 /C176C for
10 min. These primers multiplex RT-PCR products bands
for CIAP-2 and bactin were detected at 385 and 300 bp,
respectively. They were visualized by ethidium bromide
staining on 2% agarose gel. In all the runs, 1 ll of DEPC-
treated water was used instead of the RNA sample asnegative control (Fig. 2).
bactin products bands were used as reference bands for
the relative quantity of the CIAP -2RNA bands. The signal
intensities of CIAP-2 RNA band in each sample weredetermined relative to that of ß actin in the same sample
using Gel pro (3.1) Media cybernetics (USA) software to
determine the relative amount of CIAP-2 RNA in different
samples [ 19], and the result was expressed as CIAP-2/ b
actin 9100 to be expressed in the percent (%) form.
All contaminated wastes included in this study have
been sealed and discarded in strong impermeable biohazardbags for further safe transport of the theme according to
Ain Shams University hospitals’ infection control biohaz-
ard waste disposal policy.
Statistical analysisThe data were expressed as median, mean ±standard
deviation, and the chi-square analysis ( v
2) of the associa-
tion with category variables. The threshold value for
optimal sensitivity and specificity of CIAP-2 expression
with HPV-16 infection in malignant tissues was determinedby ROC (receiver operating characteristics curve) [ 20]. The
cutoff value that maximized the sum of sensitivity and
specificity was chosen for discrimination between HPV-16carcinogenicity mediated by CIAP-2 or other pathways in
malignant tissue. The specificity, positive predictive value
(PPV), negative predictive value (NPV), and accuracy werecalculated according to standard statistical methods. All
statistical analysis were performed using the software
package SPSS for Windows, version 15.0 (SPSSInc.,Chicago, IL). Significant Pvalue considered when it is
\0.05.
Results
HPV-16 positivity rate in all tissue samples
The positivity rate of HPV-16 was examined by PCR in
both groups 1 (malignant tissues) and 2 (safety margin
tissues). Twenty-six out of 30 (86.7% sensitivity) showed
positive HPV-16 bands in group 1, and only 5 out of 30(16.7%) showed positive HPV-16 bands in group 2
(specificity was 83.3% ). There was significant difference
between the two groups ( P=0.00), (Table 1).There were
no significant differences found with HPV-16 positivity
Fig. 2 ROC curve of CIAP-2 positivity in malignant tissue samples
in relation to HPV-16 positivity against HPV negative cases as
control group: The best cutoff value for discrimination between HPV-16 carcinogenicity mediated by CIAP-2 or other pathways inmalignant tissue. Area under the curve is significantly high (0.914).
Arrow denotes cutoff point at [79.1% (CIAP-2/ bactin) at which
sensitivity is 73.9% (17 true positive samples out of 23 positiveCIAP2 in the HPV infected tissues) and specificity is 100% (no truepositive samples out of 23 positive CIAP2 in the HPV free tissues)Table 1 HPV-16 positivity rate in all tissue samples
HPV Cancer tissue
(n=30)Safety margin tissue
(n=30)v2(P)
Groups n (%) 16.133
(0.00)*Positive 26 (86.6%) 5 (16.7%)
Negative 4 (13.4%) 25 (83.3%)
*Pvalue\0.05 is significant, v2chi squareMed Oncol
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among head and neck cancer patients in relation to any of
the studied clinicopathological factors ( P\0.05) as shown
in (Table 2).
CIAP-2 positivity rate and semiquantitative RT-PCR
analysis in all tissue samples
Twenty-three out of 30 tissues (76.7% sensitivity) of group
1 tissue samples showed positive CIAP-2 by RT-PCR. No
positive bands were shown in group 2 tissue samples with100% specificity with high significant difference in CIAP-2
expression between the 2 studied groups, (P=0.003 ).B y
semiquantitative RT-PCR, the mean CIAP-2 concentra-tions in both groups were measured and they showed a
statistical significant difference between the two groups
(P=0.00), (Table 3). There were no significant differ-
ences found with CIAP-2 positivity among head and neckcancer patients in relation to the different clinicopatho-
logical factors ( P\0.05) shown in (Table 4).
CIAP-2 positivity rate and semiquantitative RT-PCR
analysis in relation to HPV-16 positivity in malignant
tissues
Nineteen out of 26 (73.1%) of HPV-16 positive samples
showed positive CIAP-2 bands by RT-PCR with a signif-
icant association between CIAP-2 and HPV-16 positivity
as it was demonstrated using chi-square analysis
(P=0.01). By semiquantitative RT-PCR, there was sig-
nificant increase in CIAP-2 concentration in HPV-16
positive samples than in HPV-16 negative samples
(P=0.01) as shown in (Table 5).
The best cutoff value at 79.1% (CIAP-2/ bactin) was
calculated using ROC curve shown in (Fig. 2). That was
able to discriminate between HPV-16 carcinogenicitymediated by CIAP-2 or other pathways in malignant tissues
with high significance. The sensitivity of using this cutoff
value was 73.9% (17 true positive samples out of 23positive CIAP2 in the HPV-16 infected tissues) with
absolute specificity (no true positive samples out of 23
positive CIAP2 in the HPV-16 free tissues). The overallsensitivity, specificity, positive (PPV) and negative (NPV)
predictive values, accuracy of HPV-16 and CIAP-2 are
shown in (Table 6).
Discussion
Head and neck malignancies represent the sixth most
common malignancy worldwide and they mainly originatefrom the epithelium that lines the upper aero digestive
tract. They are arising in a multistep process resulting from
the gathering of genetic and epigenetic defects [ 1,4,21].
Sexual transmission of HPV-16 primarily through oro-
genital intercourse might be the reason for the increase in
the incidence of HPV-16-related oropharyngeal [ 14]. It is
worth mentioning that tumor HPV-16 status is stronglyTable 2 The positivity rates of HPV-16 among head and neck cancer
patients in relation to different clinicopathological factors
Clinicopathological factors (n) HPV P
Positive
(n=26)Negative
(n=4)
Sex Male (n =15) 13 (50%) 2 (50%) 1
Female (n =15) 13 (50%) 2 (50%)
Smoking Smokers
(n=20)17 (65.4%) 3 (75%) 0.75
Nonsmokers
(n=10)9 (34.6%) 1 (25%)
Pathology SCC (n =18) 15 (57.7%) 3 (75%) 0.49
ISCC (n =12) 11 (42.3%) 1 (25%)
Grade Low (1, 2)
(n=24)21 (80.8%) 3 (75%) 0.83
High (3) (n =6) 5 (19.2%) 1 (25%)
Stage Early (I, II)
(n=19)18 (69.2%) 1 (25%) 0.26
Late (III, IV)
(n=11)8 (30.8%) 3 (75%)
*Pvalue\0.05 is significant
Table 3 CIAP-2 positivity rate and semiquantitative RT-PCR analysis in all tissue samples
CIAP-2 Cancer tissues (n =30) Safety margin (n =30) v2(P)
Groups (n)
Positive 23 (76.7%) 0 (0.0%) 8.533 (0.003)*Negative 7 (23.3%) 30 (100%)
Semiquantitative RT-PCR of CIAP-2 Cancer tissues (n =30) Safety margin tissues (n =30) P
(CIAP-2/ bactin) % (Mean ±SD) 117.5 ±51.77* 0 0.00*
(CIAP-2/ bactin) % Range 48.65–181.1 0
*Pvalue\0.05 is significantMed Oncol
123
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associated with therapeutic response and survival that
increases the importance of HPV-16 diagnosis before the
therapeutic decision. It was reported by two randomizedtrials that have shown significantly improved 2-year overallsurvival in stage III and stage IV HPV-16-related oropha-
ryngeal carcinoma compared with non-HPV-16-related
cancers (87.5% and 95% compared with 67.2% and 62%,
respectively) [ 6,22–26]. This encouraged Acha-Sagredo
et al. [ 27] to try to find an easy and harmless methodology
for HPV-16 diagnosis by the detection of HPV-16 genomic
DNA in oral smears.
The association between cigarette smoking and head and
neck cancer has been known for over 30 years, and ciga-
rette smoke is a known rich source of chemical carcinogens
and reactive oxygen species, many of which likely con-
tribute to carcinogenic potential [ 14,28]. However, in the
present study, although smokers were more frequent
(66.7%) than nonsmokers (33.3%), but there were no sig-
nificant differences among head and neck cancer cases andsmoking. Our results agree with Lindel et al. [ 29] in the
lack of significant association between HPV-16 infection
and smoking. However, Gunnell et al. [ 30] suggested a
synergistic effect between smoking and both HPV-16 sta-
tus and HPV-16 viral load. These wide variations of results
among different studies depend mainly on three variables:the site of the tissue examined, the geographical distribu-
tions and the technical issues or the HPV-16 molecular
assay employed.
In our study, the relation between carcinoma and
infection with human papillomavirus-16 (HPV-16) and the
possible relation with the expression of CIAP-2 gene asTable 4 The positivity rates of CIAP-2 among head and neck cancer
patients in relation to different clinicopathological factors
Clinicopathological
factors (n)CIAP-2 P
Positive
(n=23)Negative
(n=7)
Sex Male (n =15) 10 (43.4%) 5 (71.4%) 0.195
Female (n =15) 13 (56.6%) 2 (28.6%)
Smoking Smokers
(n=20)15 (65.2%) 5 (71.4%) 0.647
Nonsmokers
(n=10)8 (34.8%) 2 (28.6%)
Pathology SCC (n =18) 12 (52.2%) 6 (85.7%) 0.205
ISCC (n =12) 11 (47.8%) 1 (14.3%)
Grade Low (1, 2)
(n=24)20 (87%) 4 (57.1%) 0.114
High (3) (n =6) 3 (13%) 3 (42.9%)
Stage Early (I, II)
(n=19)15 (65.2%) 4 (57.1%) 0.561
Late (III, IV)
(n=11)8 (34.8%) 3 (42.9%)
*Pvalue\0.05 is significant
Table 5 CIAP-2 positivity rate and semiquantitative RT-PCR analysis in relation to HPV-16 positivity in malignant tissues
CIAP-2 positivity HPV-16 positivity v2(P)
Positive (n =23) Positive (n =26) Negative (n =4) 6.6 (0.01)*
Negative (n =7) 19 (73.1%) 4 (100%)
7 (26.9%) 0 (0%)
Semiquantitative RT-PCR of CIAP-2 In HPV positive samples
CIAP-2 quantity HPV-16 Positivity P
Positive (n =19) Negative (n =4)
(CIAP-2/ bactin) % Mean ±SD 129.6 ±48.2* 59.9 ±18.9* 0.01*
(CIAP-2/ bactin) % Range 53–118 48.7–88.3
*Pvalue\0.05 is significant
Table 6 Sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy for the investigated parameters
Sensitivity
%Specificity
%Predictive
value %Accuracy
%
Pos Neg
HPV-16 (in all groups) to differentiate cancer from safety margin tissues. 86.7 83.3 83.8 86.2 85
CIAP-2 (In all groups) to differentiate cancer from safety margin tissues. 76.7 100 100 81.1 88.3CIAP-2 (In HPV-16 infected patients) to differentiate between HPV carcinogenicity
mediated by CIAP-2 or other pathways.73.9 100 82.6 100 63.3
Pospositive, negnegativeMed Oncol
123
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biomarker for HPV-16 carcinogenicity were addressed. It
was suggested by Bandala et al. [ 31] that the inhibitor of
apoptosis proteins (IAPs) is a family of anti-apoptotic
proteins that block cell death, in part, by inhibiting thedownstream portion of the caspase activation pathways that
several IAPs (including CIAP-2) act as part of a positive-
feedback loop that facilitates tumor cell survival in thepresence of TNF- a, which induces expression of CIAP-2
mRNA through stimulation of the transcription factor NF-
jB, and CIAP-2 itself can also stimulate the activation of
NF-jB; this results in a positive-feedback loop that acts
ultimately to prevent apoptosis. On the other hand, severalstudies have demonstrated up regulation of certain IAPs
with cancer development [ 11–13]. For this reason, Bandala
et al. [ 31] suggested the possible use of IAPs as early
diagnostic or prognostic markers and even as possible
therapeutic targets for malignancy treatment. In the current
study, CIAP-2 positivity rate was significantly higher in themalignant tissue group (23 out of 30 malignant tissues
showed positive CIAP-2 RNA), with no positive sample in
the safety margin tissues. These results reflect a highlysignificant association between CIAP-2 expression and
head and neck squamous cell carcinoma ( P=0.003).
However, there were no significant associations betweenthe positivity rate of CIAP-2 and any of the studied clini-
copathological factors.
Some authors suggested that CIAP-2 RNA can be asses-
sed using semiquantitative method [ 13,17]. In the present
study, CIAP-2 RNA transcript was analyzed by semiquan-
titative RT-PCR as to evaluate the usefulness of this markerin diagnosis of head and neck cancer in all tissue groups.
There was a high significant difference between CIAP-2
expression between group 1 and group 2. A significantassociation between CIAP-2 expression in the HNSC and
HPV-16 positivity was detected, as was stated by Herrero
et al.[32] that HPV-16 is highly associated with CIAP-2 by
the induction of its transcript in different malignant tissues.
In the present study, semiquantitative RT-PCR was done
to measure CIAP-2 concentration % in the malignant tissue,and after relating it with HPV-16 positivity, a significant
difference in (mean ±SD) of CIAP-2 concentration
between HPV-16 positive samples (129.6 ±48.2) and HPV-
16 negative samples (59.9 ±18.9) was found ( P=0.01).
The best cutoff value as calculated using ROC (Receiver
operating characteristics) curve was detected at 79.1%CIAP-2 expression (CIAP-2/ bactin). It was able to dis-
criminate between HPV-16 carcinogenicity mediated by
CIAP-2 or other pathways in malignant tissues. CIAP-2values [79.1% indicate that carcinogenicity of HPV-16 is
mediated through induction of CIAP-2 expression in these
tissues at which sensitivity is 73.9% (17 true positivesamples out of 23 positive CIAP2 in the HPV-16 infected
tissues). On the other hand, CIAP-2 values of \79.1%(6 false positive samples out of 23 positive CIAP2 in the
HPV-16 infected tissues) indicate mediation of carcinoge-
nicity of HPV-16 through other pathways like inhibition of
p53 or other tumor suppressor genes.
This study was among the first to evaluate the best cutoff
value at which we can discriminate between HPV-16 car-
cinogenicity mediated by CIAP-2 or other pathways inmalignant tissues. However, this study needs larger mul-
ticentric investigations to evaluate the diagnostic signifi-
cance of CIAP-2 as a marker in HPV-16 carcinogenicity
and its relation to HNSCC. This may open many fields
regarding new therapeutic strategies in management ofHNSCC to improve its prognosis.
Finally, we conclude that HPV-16 and CIAP-2 being
expressed in most of the HNSCC can be considered as anattractive point of research in many other cancer tissues.
Efforts should be directed to develop antisense vaccines
and chemical IAP inhibitors that may be useful for themanagement of cancer.
Acknowledgments This work was done by the financial support of
Faculty of medicine, Ain Shams University.
Conflict of interest None.
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