Efficacy evaluation of a test CINtec® p16INK4a in screening for cervical HPV infection

doi:10.4236/ojpm.2011.13020

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Vol.1, No.3, 154-163 (2011)

doi:10.4236/ojpm.2011.13020

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. [email protected]

Received 14 July 2011; revised 14 September 2011; accepted 21 October 2011.

ABSTRACT

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

1. INTRODUCTION

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.

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155

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

pathology.

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

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156

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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157

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.

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158

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

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159

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

reproducibility.

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

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160

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

illness.

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.

2. PROJECT RATIONALE

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

HPV.

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

phase.

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

HSIL-CIN3.

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

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161

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.

3. DESCRIPTION OF THE STUDY

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

year.

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

162

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 )

4. METHODS AND RESULTS

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).

5. ACKNOWLEDGEMENTS

Valentina Pafumi has carried out English language editing for this

article.

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