Vol.1, No.3, 154-163 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
Open Journal of Preventive Medicine
Efficacy evaluation of a test CINtec® p16INK4a in
screening for cervical HPV infection
Pafumi Carlo, Leanza V ito, Carbonaro A ntonio, Leanza Gianluca, S tracquadani o Maria Grazia,
D'Agati Alfio
Department of Obstetrics and Gynaecology University of Catania, Catania, Italy. pafumi@unict.it
Received 14 July 2011; revised 14 September 2011; accepted 21 October 2011.
We submitted 437 patients with cytological al-
terations that suggest viral infections to HPV
test. 154 patients (35.24%) resulted positive for
HPV; among these, 128 (83.11%) with a low de-
gree of infectivity, 19 (12.33%), with an average
degree of infectivity and 7 (4.54%) with a high
degree of infectivity).
Keywords: Screening; Cervical Infection; HPV Test
Cervical cancer (Figure 1) is the second type of can-
cer in terms of diffusion in women worldwide, a position
that it shares with colorectal cancer, while breast cancer
is at first place. Every year, about 400,000 new cases of
cervical cancer are diagnosed, with a higher proportion
among poorer classes, both in developing and in indus-
trialized Countries.
During the last 50 years, the standard identification
method consisted in the visual inspection of hundreds of
thousands of cells of the patient (morphology), trying to
identify slight alterations of the shape and size of the
cells and nuclei in order to identify pre-cancerous and
cancerous cells. This method is called Pap-test (Figure
2), from the name of its inventor, Dr. G. Papanicolaou
who developed it during the 40s.
In the cervical cancer, early diagnosis is fundamental
for a good prognosis and as a consequence, early identi-
fication programs try to identify people at risk and those
in the early stages of the pathology.
At least 140 million Past-tests a year are carried out
worldwide. With the implementation of prevention pro-
grams for the early detection of morphological anoma-
lies of cervical cells, the cervical cancer rate decreased
by about 70%.
Despite this great success, current identification and
early diagnosis techniques of cervical cancer boast rec-
ognized limits that determine unacceptable rates of false
negative and false positive diagnoses and high co sts.
This occurs because the current tests have a high degree
of subjectivity and do not detect the direct markers of the
pathology effectively.
Cervical cancer is the first cancer to be recognized by
the World Healthcare Organization as completely linked
to an infection [1]. Cervical cancer is indeed caused by
the genital infection from human papillomavirus (HPV)
(Figure 3).
As of today, mor e than 120 HPV genotypes that infect
mankind have been identified and among these, 40 are
associated to benign and malignant pathologies of the a nal -
Figure 1. Cervical cancer.
Figure 2. Pap test.
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
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genital stretch. The different types of HPV are indeed
distinguished in low and high risk of developing into
tumour. Low risk genotypes are associated to benign
lesions as anal-genital warts (Figures 4 and 5), while
those at high risk are associated to cervical cancer, in
addition to other tumours of the anal-genital stretch,
such as for example the cancer of the penis, vulva, va-
gina and anus.
Figure 3. HPV.
Figure 4. Perineal condyloma.
Figure 5. Anal condyloma.
The infection caused by HPV is very frequent in the
population: indeed, it is estimated that over 75% of se-
xually active women become infected during their life
with a HPV virus, with a prevalence peak in young wo-
men up to 25 years of age [2]. The natural history of the
infection is strongly related to the balance between host
and infective agent. Indeed, there are three possibilities
to develop the HPV infection: regression, persistence
and progression (Figures 6 and 7).
The majority (70% - 90%) of the infections caused by
papillomavirus is transitional because the virus is elimi-
nated by the immune system prior to develop a patho-
genic effect [2]. The persistence of the viral infection is
instead the necessary condition for the development of
the tumour. The acq uisition of a high risk viral genotype
increases the probability of persistent infection (Figures
6 and 7). In this case, pre-cancerous lesions can be de-
veloped which can then progress until transforming into
cervical cancer.
The probability of progression of the lesions is also
related to other factors, such as the high number of sex-
ual partners, smoking cigarettes, the long-term use of
oral contraceptives, and the co-infection with other se-
xually transmitted infections [2 ].
In general, the time that elapses between the infection
and the onsetting of pre-cancerous lesions is about five
years, while the latency for the onsetting of cervical
cancer can be of decades (Figure 8) [2].
For this reason, the preven tion of tumours is based on
screening programs, that allow to identify pre-cancerous
lesions and intervene prior to develop into tumour.
Recently, tests for the human papillomavirus (HPV)
have been adopted, which allow to clarify unclear and
slightly anomalous Pap-tests (the majority of which was
false positive) in women over 35 years of age. The use
of tests on HPV for this application is limited to a small
percentage of all Pap-tests. The sensitivity for detecting
the illness by HPV tests is higher than that of a Pap-test.
Nonetheless, the high infection rate due to general HPV
for this group of viruses in women (up to 25%) gives a
very low specificity (correlation of positive test with the
actual pathology) that seriously limits the potential
clinical utility of this test for the early detection of the
In addition, only few women infected with HPV will
develop clinically significant dysplastic lesions or (in
worse cases) cancer. For this reason, the result of HPV
tests is often an infection marker ra ther than a mar ker of
an actual disease.
Among the various HPV serotypes, those responsible
for a possible evolution in cervical cancer are serotypes
16 and 18.
Serotype 16 expresses diferent oncogenes, among f
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
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Figure 6. Regression, persistence and progression of HPV infection.
Figure 7. Acquisition of a high risk viral genotype.
Figure 8. T ime between infection and pre-cancerous lesions.
Openly accessible at
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which it seems that E6 and E7 (Figure 9) are of high
risk of tumour especially if expressed in basal and para-
basal cells subject to replicatio n. (Figure 10)
In order for this to take place, specific changes of the
host cell are needed, that alter the transcriptional control
of the viral genome [3,4]. The expression of viral onco-
genes in proliferating cells interferes with the regulation
of the cellular cycle and, after numerous bio-chemical
interactions, it changes the expression profiles of many
genes and/or proteins (Figures 11 and 12) [5].
These changes in the cells of basal and parabasal lay-
ers infected from HPV (Figure 13) take place only ra-
rely and therefore the tran sformation and carcinogenesis
process is fortunately only a very rare consequence of a
very common infection.
Due to this high incidence of HPV infections in the
healthy population, the ideal screening test for cervical
cancer should identify the modifications of the basal and
parabasal cells induced by the deregulated expression
pattern of viral genes; this test should at least in theory,
combine the high sensitivity of HPV tests with the high
specificity of the cytology, and therefore overcome the
Figure 9. Different oncogeneses of HPV-16.
Figure 10. HPV infection in basal and parabasal cells.
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
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Figure 11. Expression of viral oncogenes.
Figure 12. Bio-chemical interations of E7 oncogene of HPV.
limits of pap-tests and HPV tests in predicting the pres-
ence of lesions that need to be treated or anyhow moni-
tored. Being able to predict the ev olution of an illness is
also one of the major challenges of modern medicine.
High risk HPV are able to contribute to the development
of the malignant phenotype by means of numerous
strictly inter-related mechanisms. Since these molecular
interactions are mediated by proteins, the logic strategy
is to “dissect’’ the complex molecular path and study
these markers, using a variety of research techniques
(Figure 14).
In addition to predict the trend of the illness and viral
events, the effective and specific molecular markers
should not be dependent on the many technical limita-
tions of the current technology for the diagnosis of cer-
vical cancer and its precursors, which are mainly based
on the morphological interpretation of sample cells of
th cervix uteri, invalidated by the subjectivity of the e
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
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Figure 13. Basal and parabasal cells in fected by HPV.
Figure 14. Modulation of viral transcription by host cell promoters.
Cytopathologist and therefore, by the low inter-observer
The identification of cellular modifications associated
to the deregulated expression of viral oncogenes E6 and
E7 in basal and parabasal cells can add very significant
diagnostic information.
Therefore, the scientific community is focusing its at-
tention on new markers, on which work standardization
and clinical validation, the diagnostic methods will de-
pend in the following years. Ideally, a marker used in the
clinical practice should be cost-effective, easy to iden tify
in sampled biological material with non-invasive meth-
ods, of high sensitivity and high specificity and be
adaptable to automated technologies that can manage a
large volume of work and guarantee high reproducibility.
Recent studies allowed to identify a biomarker, pro-
tein p16, which is hyper-expressed in cervical dysplastic
cells and which hyper-expression is directly linked to a
clear activity of the viral oncogen e E79 (Figure 15) [6].
P16INK4a is the biomarker on which mtm laboratories is
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
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Figure 15. Protein P16.
focusing with its patented products to develop diagnostic
tests sensitive to cervical cancer. The inhibitor of the cy-
clin-dependent kinase shows a significant hyper- expres-
sion in the cancerous and pre-cancerous tissue, that
makes it a suitable candidate to be a bio-marker of the
As known, the cervical cancer is caused by a persis-
tent infection of human papillomavirus at high risk
(HR-HPV). The hyper-expression of p16INK4a is linked to
the oncogenic transformation caused by a persistent in-
fection of HR-HPV. In any case, differently from the
detection of the simple presence of HR-HPV, the identi-
fication of the hyper-expression of p16INK4a shows the
inactivation of the control of the cellular cycle mediated
by the oncoproteins of HR-HPV or the main pathologi-
cal process in cervical cancer.
In normal conditions, p16 is an oncosuppressor that
regulates the cellular cycle, interrupting the transition
signal of the cell from phase G1 to phase S, phases dur-
ing which the cell synthesizes the proteins, then it repli-
cates the DNA.
P16 acts by inhibiting cyclin-dependent kinases re-
sponsible for the phosphorylation of Retinoblastoma
protein (pRb) and therefore the transcription of factor
E2F that, by regulating the production of specific pro-
teins of phase S, it allows the cell to proceed from one
phase to the other. The synthesis of p16 is regulated by a
negative feedback mechanism with the same factor E2F.
The main oncogenic activity of E7 is to prevent the
function of pRB. This way pRB does not bind to tran-
scription factor E2F, determining the transcription of
genes that promote cellular proliferation. Only in trans-
forming HPV infections in which the oncogenic process
has begun, the levels of protein E7 are generally high in
replication-competent cells. For this reason p16INK4a is a
more accurate predictor of cervical cancer compared to
the presence of HR-HPV.
The hyper-expression of protein p16 points out there-
fore an alteration of the cellular cycle by oncogene E7
with the increase of the DNA synthesis and block of
cellular differentiation, thus inducing a greater probabil-
ity for the cell to develop into tumour.
Since p16INK4a is a cellular protein, it can act as bio-
marker, independent from the type of individual
HR-HPV that indicates the cervical cancerous patho-
logical process in progress. There are many types of
HR-HPV, but in any case the effect of oncoproteins E7 is
the same in blocking the pRB and leads to the hy-
per -e xp ression of p16INK4a.
The hyper-expression of p16INK4a is a direct marker
of the oncogenic activity of all various types of high risk
The presence of HPV does not mean that the patient
will certainly develop a cervical cancer in the future. The
frequency in younger woman may reach even 30%. The
exam of the HPV is therefore scarcely useful in identi-
fying the illness in young women. Instead p16INK4a is
expressed only in the oncogenic process of the cervical
cancer and is not more prevalent in young women.
In order to selectively identify the p16INK4a in the cer-
vical tissue, the diagnostic kits of mtm laboratories use
the clone of the patented antibody E6H4TM which is
highly selective and sens itive to the presence of p16INK4a.
The objective that we aim to pursue with this project
is to analyse this emerging marker recently proposed
(2007) by mtm laboratories, that has already passed the
first experiments, it has been introduced inside the kit,
and it is currently undergoing the clinical validation
The role of p16 as marker of cervical dysplasias in
histological sections [7], traditional cytological imprints
(past-test) [8] and th in layer [9] has been proven by now.
In a study conducted at the Cytology Division of Perugia,
a method to search for p16 on conventional pap-tests
was used, and it was observed that with the increase of
the seriousness of the lesions, the positivity of p16 in-
creases in percentage, until reaching 100% in cases
The study conducted at Istituto Tumori Regina Elena
also shows a statistically significant inter-relation (k =
0.81) between hyper-expression of p16 and high risk
HPV infection (HR-HPV), identifying p16 as specific
and sensitive biomarker of the active expression of on-
cogene E7.
The detection of a small percentage of high degree in-
tra-epithelial lesions (HGCIN) in patients with light cy-
tological anomalies (ASCUS/LSIL) is a significant pro-
blem for cytological screening (Figure 16).
For this reason, different studies have been conducted
to evaluate the efficacy of p16 as marker able to identify
patients with HGCIN among those with ASCUS or LSIL
on cytological anomali es. In particular, a study has found
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
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Figure 16. High-grade intra-epitelial lesions.
a specificity and sensitivity for p16 in ASCUS group of
95% and 84%, and of 100% and 81% respectively in
LSIL group, suggesting that the use of p16 in addition to
the cervical cytology for the triage of patients with AS-
CUS/LSIL allows identification with a good degree of
sensitivity and specificity [10].
The management problem of ASCUS/LSIL discussed
in ALTS studies (ASCUS/LSIL Triade Study for Cervi-
cal Cancer) indicated that 83% of women with a cyto-
logical diagnosis of LSIL were positive to HPV test and
that no more than 25% of these women were developing
a high degree lesion. The same was occurring for AS-
CUS, indeed a strategy that was foreseeing the HPV test
was more sensitive in detecting serious cervical lesions,
but the specificity was much lower[11].
In a recently published study [11] a role of p16INK4a
was studied, as potential complementary marker for cy-
tological diagnose, using CINtec® p16INK4a Cytology
Kit (Dako, Glostrup, Denmark) to evaluate the expres-
sion of p16INK4a on cytological imprints on thin layer and
compare it with the results of HR_HPV(hc2) test. The
results obtained showed the higher diagnostic specificity
of CINtec® 16INK4a assay compared to hc2 in detecting
high degree lesions.
1) The diagnostic and prognostic meaning that this
protein has, associated to the fairly easy method of de-
tection and low cost, triggered our interest for p16INK4a
as useful biomarker in addition to traditional morphol-
ogy to improve the quality of the cytological diagnose
and to select lesions that, despite appearing slight during
a morphological test, could progress into more severe
lesions and therefore they must be followed up closer
and eventually be treated. This would avoid useless col-
poscopies, anxiety and reduction of healthcare costs.
In particular, using p16 in combination with the Pap
test as additional useful instrument to clarify difficult
cases, such as:
Cases of ASC--US
Cases of LSIL
Cases of ASC--H
Repetition of the cytology
Glandular anoma lies
in order to significantly decrease the number of patients
treated erroneously or without any need.
2) Evaluate the efficacy of this new method, CINtec®
p16INK4a Cytology Kit, on our population under study,
through an analysis of data input in the database of the
Pathological Anatomy Service of Ospedale S.Bambino
in Catania.
All the women of any age that will come to the Divi-
sion of Gynaecology and Obstetrics of Ospedale S.
Bambino from January 2009 to 31 December 2009 will
be enrolled in our study.
After collecting anamnestic data, a cervical-vaginal
smear will be taken from each of these women, that will
be subject to reading at the microscope by the special-
ized staff of the Pathological Anatomy Service.
In the cases indicated in point (2), a second test will
be carried out on the smears to evaluate the immu-
nopositivity of p16 using the CINtec® p16INK4a Cy-
tology Kit of mtm laboratories, in order to better manage
the patients, especially those with ASCUS/LSIL.
In case the p16 test is positive, these cases will be sub-
ject to a closer follow-up (from 3 to 6 months) in case of
ASCUS and to colposcopy in case of LSIL, while in
case the test is negative, the follow-up will be after one
CINtec® Cytology Kit (mtm)
The CINtec® Cytology Kit is a qualitative immuno-
cytochemical test for the evaluation of the hyper- ex pressed
inhibitor of cyclin-dependent kinase, the p16INK4a protein,
on cervical cytological samples. P16INK4a is a biomarker
that indicates directly the oncogenic activity of the high
risk human papillomavirus (HR-HPV) responsible for
the onsetting of cervical cancer. This is a much more
precise instrument for the diagnosis of a cervical cancer
compared to traditional technologies.
The cytological test CINtec® is based on the clone
f antibody E6H4TM developed specifically as method o
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
Copyright © 2011 SciRes. http://www.scirp.org/journal/OJPM/
Figure 17. HPV lesions.
(a) (b)
Figure 18. Viral protein in cervic al cancer.
Table 1. Results.
Patients HPV pos HPV neg Low infectivityMedium infectivity High infectivity
437 283 (68%) 154 (32%) 128 (83%) 19 (12%) 7 (5%)
to identify p16INK4a in cytological samples. The sensitiv-
ity and specificity of the clone of antibody E6H4TM was
verified and proven by more than 50 clinical studies. It is
important to underscore that E6H4TM does not show
crossed reactivity with Trichomonas (a protozoal infec-
tion of the vagina) that renders other potential antibodies
unacceptable since they are responsible for too many
false positive results. The components of the kit, pro-
duced according to GMP in combination with the opti-
mized antibody, ensure quality and reproducible results
in the evaluation of a large range of biolog ical samples.
Openly accessible at
TheCINtec® Cytology test was developed th rough th e
immunocytochemical coloration of cervical cytological
imprints. It was approved for use in:
Cytological imprints in liquid phase (the cells col-
lected from the cervix are re-suspended in a fixative
liquid with alcoholic base, and then cytological im-
prints in thin layer are prepared.
Traditional cervical smears (the cells are directly
transferred from the sampling device to the slide,
then fixed )
We subject 437 patients with cytological alternations
that suggest viral infections, to HPV test. 154 patients
(35.24%) resulted positive for HPV; among these, 128
(83.11%) with a low degree of infectivity, 19 (12.33%),
with an average degree of infectivity and 7 (4.54%) with
a high degree of infectivity) (Table 1).
Valentina Pafumi has carried out English language editing for this
[1] IARC Working Group (1995) IARC Monographs on the
evaluation of carcinogenic risks to humans. International
Agency for Research on Cancer, Lyon, 64.
[2] Frazer, I.H., Cox, J.T., Mayeaux, E.J., Franco, E.L., et al.
(2006) Advances in prevention of cervical cancer and
other human papillomavirus-related diseases. Pediatric
Infectious Disease Journal, 25, S65-S81.
[3] Munger, K., et al. (1989) The E6 and E7 genes of the
human papillomavius type 16 together are necessary and
sufficient for transformation of primary human keratino-
cytes. Journal of Virology, 63, 4417-4421.
[4] Zur-Hausen, H., de Villiers, E.M. (1994) Human papil-
lomaviruses. Annual Review of Microbiology, 48, 427-
447. doi:10.1146/annurev.mi.48.100194.002235
P. Carlo et al. / Open Journal of Preventive Medicine 1 (2011) 154-163
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
[5] Zur-Hausen H., (2000) Papillomaviruses causing cancer:
Evasion from host-cell control in early events in car-
cinogenesis. Journal of the National Cancer Institute, 92,
690-698. doi:10.1093/jnci/92.9.690
[6] Von Knebel-Doeberitz, M. (2002) New markers for cer-
vical dysplasia to visualize the genomic chaos created by
aberrant oncogenic papillomavirus infections. European
Journal of Cancer, 38, 2229-2242.
[7] Benevolo, M., et al. (2006) Immunohistochemical ex-
pression of p16INK4a is predictive of HR-HPV infection in
cervical low-grade lesion. Modern Pathology, 19, 384-
391. doi:10.1038/modpathol.3800551
[8] Passamonti, B., et al. (2006) P16: A method for directed
research on abnormal convenzional pap-test. Pathologica,
98, 417.
[9] Bibbo, M., et al. (2002) Procedure for immunocyto-
chemical detection of p16INK4a antigen in thin-layer, liq-
uid-based specimens. Acta Cytologica, 46, 25-29.
[10] Nicolas, W., et al. (2009) Triage of women with ASCUS
and LSIL cytology. Cancer Epidemiol Biomarkers Pre-
vention, 18, 1341-1349.
[11] Meyer, J.L., et al. (2007) Evalution of p16INK4 expression
in thin-prep cervical specimens with the CINtec® 16INK4a
assay. Cancer Cytopathology, 11, 83-92.