Journal of Clinical Virology 56 (2013) 69–71

Contents lists available at SciVerse ScienceDirect

Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Short communication

Cervical biopsies and cytological smears – A comparison of sample materials in

HPV diagnostics
Christine Webersinke a,∗ , Stefan Doppler a,c , Franz Roithmeier b,d , Wolfgang Stummvoll b,e , Rene Silye a,f
a
Institut für allgemeine Pathologie und Neuropathologie, LNK Wagner-Jauregg, Wagner Jaureggweg 15, A 4020 Linz, Austria
b
Abteilung für Gynäkologie, Krankenhaus der Barmherzigen Schwestern Linz, Seilerstätte 4, A 4010Linz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Background: Cervical cancer is causally related to cervical infections by oncogenic human papillomavirus
Received 14 August 2012 (HPV) genotypes. To improve the quality of diagnosis evaluation of screening methods and their HPV
Accepted 24 September 2012 type detection rate is an important part for this item.
Objectives: Two different cervical specimens of the same patients were analysed simultaneously with
Keywords: molecular HPV subtyping methods to find the most sensitive sample material for cervical cancer
Human papilloma virus
screening.
Molecular subtyping
Study design: Biopsy specimens and cytological smears of the cervix of 443 patients were analysed for
Comparison
Cytological smear
human papilloma virus (HPV) subtyping by a macroarray from Chipron, Germany, which allows a dif-
Cervical biopsy ferentiation of 16 high and 16 low risk types. Results were compared for reliability and differences were
Macroarray Chipron studied.
Results: Both sample material groups showed HPV conformity of 70%, 23% more subtypes could be
detected in smears in contrary to biopsies but only 6% vice versa. 14 biopsies and 7 smears were HPV
negative although the concerning second sample type of the patients was HPV positive. HPV 16 as one
of the most relevant subtypes in cervical cancer pathogenesis was missed in the biopsies’ group with
34.3% out of 35 HPV 16 positive smear cases, whereas only one smear failed to discover this subtype
contrariwise.
Conclusion: Comparison of the examination results shows that subtyping of smear samples is able to
detect more subtypes than by biopsy specimens. The probability to underdiagnose HPV 16 and to get a
false negative result in bioptic sample material favours smear as method of choice for HPV subtyping.
© 2012 Elsevier B.V. All rights reserved.

1. Background distribution and/or statistic evaluation of co-infections in detail,

Cervical cancer is causally related to cervical infections by a
set of well characterised oncogenic human papillomavirus (HPV)
genotypes. Cervical cancer screening is based on colposcopy and
cytological evaluation of cervical smears (i.e., cervical cytology or
PAP smears) and there is an ongoing debate about how to include
HPV-testing in the screening algorithm.1 Current official Austrian
guidelines recommend HPV testing in case of PAP III.2
HPV and cancer pathogenesis are the subject of numer-
ous papers discussing regionally and age related HPV subtype
∗ Corresponding author. Tel.: +43 505546226314; fax: +43 505546226304.
E-mail addresses: christine.webersinke@gespag.at (C. Webersinke),
stefan.doppler@gespag.at (S. Doppler), franz.roithmeier@bhs.at (F. Roithmeier),
wolfgang.stummvoll@bhs.at (W. Stummvoll), rene.silye@gespag.at (R. Silye).
c
Tel.: +43 505546226308; fax: +43 505546226304.
d
Tel.: +43 73276774336; fax: +43 73276777676.
e
Tel.: +43 73276774829; fax: +43 73276777676.
f
Tel.: +43 505546226300; fax: +43 505546226304.
yet.3 But none of them pay attention to the question which spec-
imen is best for analysing. Another very important issue is the
evaluation of screening methods and their HPV type detection rate.
Early papers matched cytological smears and biopsies by using
in situ hybridisation and dot blot analysis but not by DNA amplifi-
cation methods. Therefore only few subtypes were covered in these
studies.4–7
2. Objectives
Here we try to contribute to the issue, whether type-specific
HPV-DNA present in biopsies with conspicuous pathology corre-
sponds in cervical smears of the same patient, to identify the most
sensitive winning method for an efficient cervical cancer screening.
3. Study design
Biopsy specimens (B) and cytological smears (S) of 443 patients
with PAP and/or CIN pathology from the hospital Barmherzige

1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jcv.2012.09.008

Descargado para Anonymous User (n/a) en Universidad Nacional Autonoma de Mexico de ClinicalKey.es por Elsevier en junio 27, 2018.
Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2018. Elsevier Inc. Todos los derechos reservados.
70 C. Webersinke et al. / Journal of Clinical Virology 56 (2013) 69–71

Fig. 1. Photo of an agarose gel electrophoresis: human housekeeping genes are shown as 100 bp, 200 bp, 300 bp, 400 bp, 600 bp amplificates on the left,9 both HPV products
are shown on the right, mix “125” prime ∼175 bp and MY 11/MY09 ∼450 bp products. Listed numbers represent diagnostic probes in double (x/1 and x/2). Patients 1114 and
1117 are HPV positive, 1115, 1116 and 1125 are HPV negative, whereas 1115 and 1116 generate unspecific human genomic DNA byproducts; ladder = 100 base pair ladder
(Invitrogen, Germany).

350 this group 41% exhibit no HPV, 36% single infections, 14% double,
310 (70%)
300 6% threefold and the rest up to 7-fold infections.
Additional subtypes could be detected in 100 cytological smears
250
number (percent)

(S > B) but only in 28 biopsies in comparison to the respective coun-

200
150
100 (23%)
100
28 (6%)
50
5 (1%)
0 Analysing the variable results of both sample materials in terms
S=B S>B S<B S≠B
smears (S) versus biopsy specimens (B)
Fig. 2. Comparison of HPV subtyping in 443 cytological smears and biopsy speci-
mens: S = B number and subtypes are identical, S > B more subtypes in smears, S < B
more subtypes in biopsies, S =
/ B subtypes in common but additional subtypes in
both sample materials.
Schwestern (Linz, Austria) were collected in a 4 years period
from November 2007 to December 2011 and analysed simulta-
neously for HPV subtyping using the macroarray HPV 3.5 from
Chipron, Germany. DNA preparation was performed on an auto-
mated nucleic acid purificator – QIAsymphony instrument (Qiagen,
Germany) – using QIAsymphony Virus/Bacteria Mini Kit (Qia-
gen, Germany). Previously, a 1-h digestion with proteinase K in
ATL buffer (Qiagen, Germany) was done to break up the tis-
sue. Two amplifications were performed using primermix “125”
– an equimolar 30 primerpair mixture containing forward primers
located nearby MY11 sequence and reverse primers equally to GP6+
– and primers MY11/MY09 and HotStarTaq DNA Polymerase (Qia-
gen, Germany). This two-fold PCR allows for a better detectability
of 16 high and 16 low risk subtypes.8 Both PCR products were
mixed and hybridisation was done according to the manufacturer’s
instructions (Chipron, Germany). Since there is no internal control
in the kit, DNA quality control was done with a PCR of 5 house-
keeping genes.9 All PCR amplificates were visualised by agarose
gelelectrophoresis (Fig. 1).
4. Results
Comparing the results of both samples smears and biopsies a
high degree of conformity was seen (Fig. 2). 310 of 443 patients
showed an identical number and subtyping of HPV (S = B). Within
terpart sample material (S < B). Most of them contained only a single
subtype more (∼83%), the rest two (∼12%) or more. The typing
results of 5 women with multiple HPV infection showed a stock
of conforming subtypes but both parallel tested sample materi-
als detected an additional type (S = / B). So they have no critical
influence in this study (Fig. 2).
of high risk HPV, there is a greater relevance of overlooking cancer
risk in biopsy specimens than in cytological smears. 22% of biop-
sies which contained less HPV subtypes than smears (S > B) missed
a high risk type in comparison to the simultaneously obtained cer-
vical swab, but only 19% contrariwise (S < B).
HPV 16 is one of the most relevant subtypes in cervical cancer
pathogenesis. Biopsies missed it in 13.6% of all 100 samples, or in
34.3% out of 35 HPV 16 cases (S > B), whereas only one smear failed
to discover this subtype (S < B). In this single case HPV 16 positivity
was also extremely weak in the corresponding biopsy specimen.
14 cervical biopsies were HPV negative, whereas the correspon-
dent swabs were positive (S > B). The opposite constellation was
only seen in 7 cases (S < B). Measurement of DNA and quality PCR
control by 5 housekeeping genes9 revealed no difference in DNA
quality of the matching sample materials. This finding could be the
result of inappropriate sample collection.
5. Discussion
As a conclusion, the probability to underdiagnose HPV 16 and
to get a false negative result in bioptic sample material favours
smear as method of choice for HPV subtyping. Moreover, examina-
tion of smears yields more subtypes than biopsies. Current medical
opinion is that infection with multiple high risk subtypes has no
influence on higher cancer risk. Detailed subtyping in screening
programs is not performed routinely though, so there are few
studies yet. The polymorphic human histocompatibility leukocyte
antigen (HLA) I and II as well as killer immunoglobulin-like receptor
(KIR) genes could be responsible to confer protection or suscepti-
bility in high grade CIN development.10 So, knowledge about the
number of subtypes could direct to a disadvantaged HLA constel-
lation.

Descargado para Anonymous User (n/a) en Universidad Nacional Autonoma de Mexico de ClinicalKey.es por Elsevier en junio 27, 2018.
Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2018. Elsevier Inc. Todos los derechos reservados.
 C. Webersinke et al. / Journal of Clinical Virology 56 (2013) 69–71 71

At last it should be stated that cervical biopsies are good sam- 2. Breitenecker G, Girardi F, Joura EA, Kohlberger P, Reich O, Widschwendter A,
ple materials in routine HPV diagnosis, too. Diagnosis is justified if et al. Leitlinie für die Diagnose und Therapie von Cervikalen Intraepithelialen
Neoplasien (CIN) und Mikrokarzinomen der Cervix uteri. Arbeitsgemeinschaft
there is a clinical consequence. für Kolposkopie und Arbeitsgemeinschaft für Gynäkologische Onkologie in der
ÖGGG. Speculum 2005;23:20–5.
Funding 3. Rossi PG, Bisanzi S, Paganini I, Di Iasi A, Angeloni C, Scalisi A, et al. Prevalence
of HPV high and low risk types in cervical samples from the Italian general
population: a population based study. BMC Infect Dis 2010;10:214–26.
None. 4. Auvinen E, Hukkanen V, Lehmijoki J, Salmi T, Arstila P. Comparison of smear
specimens with biopsy specimens in a nucleic acid hybridization test for human
papilloma virus (HPV) infection. Acta Obstet Gynecol Scand 1989;68(7):627–31.
Competing interests 5. Gitsch G, Reinthaller A, Tatra G, Breitenecker G. Diagnosis of cervical intraepithe-
lial neoplasia and human papillomavirus infection: punch biopsy versus cervical
None declared. smear. Arch Gynecol Obstet 1991;249(4):179–84.
6. Hukkanen VI, Auvinen E, Salmi T, Virtanen M, Kujari HP. A comparison of human
papillomavirus detection rates by dot blot assay from smear and biopsy speci-
Ethical approval mens with regard to human papillomavirus type and histologic diagnosis. Am J
Clin Pathol 1994;101(6):694–7.
7. Troncone G, Herrington CS, Cooper K, de Angelis ML, D’ McGee JO. Detection
Not required. of human papillomavirus in matched cervical smears and biopsy specimens by
nonisotopic in situ hybridization. J Clin Pathol 1992;45:308–13.
Acknowledgment 8. Weimin QU, Jiang G, Cruz Y, Chang C, Ho GY, Klein R, et al. PCR detection
of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+
primer systems. J Clin Microbiol 1997;35:1304–10.
We thank Jennifer Kaar for statistical preliminary work. 9. Van Dongen J, Langerak A, Brüggemann M, Evans P, Hummel M, Lavender F,
et al. Design and standardization of PCR primers and protocols for detection
of clonal immunoglobulin and T-cell receptor gene recombinations in suspect
References lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT98-
3936. Leukemia 2003;17:2257–317.
1. Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD. Natural history of cervicov- 10. Snijders PJ, Steenbergen RD, Heideman DA, Meijer CJ. HPV-mediated cervical
aginal papillomavirus infection in young women. N Engl J Med 1998;338:423–8. carcinogenesis: concepts and clinical implications. J Pathol 2006;208:152–64.

Descargado para Anonymous User (n/a) en Universidad Nacional Autonoma de Mexico de ClinicalKey.es por Elsevier en junio 27, 2018.
Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2018. Elsevier Inc. Todos los derechos reservados.