Cervical cancer screening: which HPV test should be used—L1 or

European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 45–46
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and
Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb
Expert opinion
Cervical cancer screening: which HPV test should be
used—L1 or E6/E7?
W.A.A. Tjalma a,*, C.E. Depuydt b
a
Department of Gynecology, Multidisciplinary Breast and Gynecologic Oncology Clinic, Antwerp University – Hospital, University of Antwerp, Antwerpen,
Belgium
b
Department of Molecular Pathology (RIATOL), Sonic Healthcare Benelux, Antwerpen, Belgium
A B S T R A C T
Article history:
Received 9 March 2013
Received in revised form 19 June 2013
Accepted 23 June 2013
Cervical cancer can and should be a historical disease. The reality, however, is that every year more than
half a million women are diagnosed with cervical cancer and a quarter of a million die of this disease. The
causal factor for cervical cancer is a persistent HPV infection and therefore a vaccine was developed:
prophylactic HPV vaccination will reduce cervical cancer by 70%. Screening based on cytology will miss
more than 40% of the abnormalities. The introduction of vaccination should lead to the reintroduction of
cervical cancer screening based on HPV detection. Primary HPV screening followed by cytology will
detect almost all abnormalities. Not all HPV tests, however, are the same! Clinicians are generally not
aware that there is a huge difference among HPV tests. If a low grade lesion progresses to a high grade or
invasive cancer, their HPV is likely to integrate. During integration L1 expression can be lost, but E6/E7
expression will always remain present. If the viral HPV is completely integrated then a L1 test looking for
only L1 expression will miss this (pre)cancer, while the E6/E7 test will not miss it. HPV tests used in
cervical cancer screening should be based on the early (E) and the late (L) genes in order not to miss the
abnormality.
ß 2013 Elsevier Ireland Ltd. All rights reserved.
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Keywords:
Cervical cancer screening
HPV test
L1
E6
E7
Integration
Missing
Failure
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A R T I C L E I N F O
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Future cervical cancer screening will be based on HPV testing.
Among HPV tests there is a huge difference regarding the
choices of primers to use. Clinicians hardly know the differences
because they assume that every test is the same. Is this a
surprise? No! Did you ever bother which kit was used to
measure CA-125? This article is written to point out how
important it is to have knowledge about the different HPV tests.
For some of us the ﬁrst part could be boring, but the second half
will undoubtedly wake you up and make you grab the phone to
call the laboratory.
HPV infection is the causal factor for the development of
cervical cancer. Once infected there will be the development of a
low grade or a high grade lesion. The majority of the lesions (80%)
will disappear within two years. Low grade lesions generally
represent productive HPV virion producing infections with a
relatively low risk of progression, while high grade lesions
represent an HPV-transformed cervical cell clone with a 31% risk
* Corresponding author at: University Hospital Antwerp and University of
Antwerp, Wilrijkstraat 10, 2650 Edegem, Belgium; Tel.: +0032 3 821 59 04;
fax: +0032 3 828 39 85.
E-mail address: [email protected] (W.A.A. Tjalma).
of progression to cancer [1]. Clinically, only the lesions that
progress are of interest.
The key players in the malignant transformation are the viral
oncogenes E6 and E7, where E stands for early expression. Loss of
the transcriptional control system leads to high levels of viral
oncogene expression throughout the epithelium. This deregulation
of the viral oncogene expression and host genomic mutation is
seen in cells in which truncated viral genomes are integrated into
the host DNA [2]. The percentage of integration increases from CIN
to invasive cancer. In benign HPV lesions and CIN 1 you will ﬁnd no
integrated or episomal HPV DNA, while in HPV 16, HPV 18 and HPV
45 cancers there is up to 55–80%, 83% and 92–100% integration
respectively [2–4].
During an HPV infection, the viral genome can be present in
three different forms, episomal HR-HPV, integrated HR-HPV or in a
mixed form with integrated and episomal HR-HPV. Currently the
two most used methods for HPV DNA detection is: hybridization
(e.g. Hybrid Capture II system, HC2, Digene Corp) and PCR (e.g.
GP5+/6+, Roche, Abbott). The HC2 test was until recently the only
HPV test approved by the U.S. Food and Drug Administration. This
test uses a whole genome probe to detect 13 HR HPV types. Its
disadvantages are that it is not type-speciﬁc and that there is crosshybridization with other HPV types, leading to a lower sensitivity
and speciﬁcity than PCR.
0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejogrb.2013.06.027
16/04/2014
W.A.A. Tjalma, C.E. Depuydt / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 45–46
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Clinicians are overwhelmed with the numerous available
HPV tests. It is therefore important to know what the differences
are between an L1 test and an E6/E7 test. What is happening
when a woman gets infected with HPV? Some women will
develop a persistent HPV infection with the development of a
low grade lesion. A few of these low grade lesions will progress
to a high grade or cancer. During progression from a low grade
lesion to cancer there is often an integration of HPV in the
genome. During integration L1 expression can be lost, but E6/E7
expression remains always present, since E6/E7 are pivotal in
the development of cancer (L1 negative cancers exist, but not E6
or E7 negative cancers). In other words, if one were to use an E6/
E7 test all cancers would be detected, including the ones where
L1 was lost.
This theoretical idea is supported by a study in high grade
cervical lesions which used a test located in the L1 region (Roche
reverse line blot assay) and compared this with type-speciﬁc PCR
for HPV16 E6/E7 and HPV 18 E6/E7 [10]. Calculation reveals that
for HPV 16, 0.3% of the CIN 2 and 3.94% of the CIN 3 were L1
negative but positive for E6/E7. The same appears to be true for
HPV 18, but no precise ﬁgure can be calculated because the
percentage of HPV 18 determined by the Roche reverse line assay
is not given separately in the article. The global difference
between HPV 16 and HPV 18 tested by L1 and E6/E7 is
respectively 91.7% and 72.1%. This means that 8.3% of HPV 16
and 27.9% of HPV 18 are missed by the L1 test. The high amount
of HPV 18 is logical because HPV 18 is more often integrated. At
present we have identiﬁed four women who died of cervical
cancer and multiple women with high grade lesions who are HPV
negative for L1 and HPV positive for E6/E7. We believe it is
difﬁcult to defend an L1-only test.
References
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HPV detection with PCR can be done using either consensus
PCRs mostly detecting a conserved region in L1 (MY9/11, SPF10,
GP5+/6+) allowing the detection of many different HPV types in
one reaction, or with type-speciﬁc PCRs which can detect
individual HPV types. The L1 gene codes for the L1 capsid protein,
where L stands for late expression. Besides identifying the exact
HPV type, type-speciﬁc PCR’s are also region-speciﬁc, targeting a
speciﬁc region in the HPV genome (e.g. L1, E1/E2, E6/E7).
Considering the importance of each of these regions in cervical
cancer, the choice of which PCR-based assay will be used is crucial.
Analysis of the L1 region is done with consensus primers like
GP5+/6+, MY09/11, and SPF10. Each of these consensus PCRs
ampliﬁes a different length within the L1 region, ranging from
455 bp with MY9/11 to <100 bp fragments with SPF10. Identiﬁcation of different HPV types is then possible by hybridization of the
amplicons with type-speciﬁc probes or by DNA sequencing. A
disadvantage is that in samples with multiple HPV types, the
consensus PCR’s will not amplify (detect) all HPV types with the
same efﬁciency, and HPV types can be missed [5], making it less
usable for genotyping of multiple infections in clinical practice.
Despite the fact that the both primer GP5+/6+ and MY09/11 target
the L1 region, the MY09/11 primers detect double the amount of
multiple infections [5]. The PGMY09/11 primer was made as a
shorter more advanced version of the MY09/11. Rather surprisingly, the sensitivity of both primers is the same and the MY09/11
has a better detection of several important HPV types, including
HPV16 [6]. The commercially available Roche Linear Array HPV
genotyping test is based on the combination of the PGMY09/11
primer with a line blot assay. The SPF primer is technically
analogous to PGMY09/11, but has higher HPV detection rates than
those of MY09/11 [7]. Analyses in the E6/E7 region are done by
type-speciﬁc PCR assays and target speciﬁc sequences of viral early
genes, usually E6 and E7. The application of HPV type-speciﬁc PCRs
allows immediate discrimination between different HPV types
together with their viral load.
A comparison between MY09/11 consensus PCR and typespeciﬁc E6/E7 HPV PCRs showed that consensus PCR targeting L1
failed to detect 10.9% of HPV infections [8]. The clinical relevance of
the HPV infections missed by MY09/11 PCR was reﬂected in the
fraction of cases with cytological abnormalities and in follow-up,
showing 25.4% CIN2+ cases. The MY09/11 false negativity could be
the result of poor sensitivity, mismatch of MY09/11 primers or
disruption of L1 target by HPV integration or DNA degradation.
Furthermore, MY09/11 PCR lacked speciﬁcity for oncogenic HPVs.
Diagnostic accuracy of the PCR systems, in terms of sensitivity
(MY09/11 PCR: 87.9%; type-speciﬁc PCRs: 98.3%) and speciﬁcity
(MY09/11 PCR: 38.7%; type-speciﬁc PCRs: 76.14%), and predictive
values for histologically conﬁrmed CIN2+, suggest that typespeciﬁc PCRs should be used in a clinical setting as a reliable
screening tool with excellent cost-effectiveness and turnaround
times [8]. Another advantage of quantitative real-time PCR is the
determination of genotype speciﬁc viral load. Based on the HPV
type a risk assessment can be done and if you combine this with the
alteration or not in viral load you can have a good assumption
whether there is clearance of infection or persistent infection with
the risk of integration and progression [9].
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