FULL PAPER

British Journal of Cancer (2014) 110, 1561–1570 | doi: 10.1038/bjc.2014.32

Keywords: vaginal cancer; prognosis; HPV; p16; Ki-67

Human papillomavirus, p16INK4A, and Ki-67 in

relation to clinicopathological variables and
survival in primary carcinoma of the vagina
K Hellman*,1, D Lindquist2, C Ranhem3,4, E Wilander5 and S Andersson4
1
Gynecological Oncology, Department of Oncology, Radiumhemmet, Karolinska University Hospital, 17176 Stockholm, Sweden;
2
Oncology Research Laboratory, Department of Radiation Sciences, Umeå University, 901 87 Umeå, Sweden; 3Department of
Obstetrics and Gynecology, Västerås Centrallasarett, 721 89 Västerås, Sweden; 4Division of Obstetrics and Gynecology,
Department of Women’s and Children’s Health, Karolinska Institutet, Elevhemmet H2:00, Karolinska University Hospital Solna,
17176 Stockholm, Sweden and 5Departments of Genetics and Pathology and Clinical Pathology and Cytology, Rudbeck
Laboratory, Uppsala University Hospital and Uppsala University, 751 85 Uppsala, Sweden

Background: This study aimed to determine human papillomavirus (HPV) status and to investigate p16INK4A and Ki-67 expression
and their correlation with clinical parameters and survival in women with primary carcinoma of the vagina (PCV).

Methods: The presence of HPV DNA was evaluated by PCR. Genotyping was performed by Luminex in 68 short-term (p2 years)
and long-term (X8 years) PCV survivors. p16INK4A and Ki-67 expression was evaluated by immunohistochemistry.

Results: Human papillomavirus DNA was detected in 43% of patients, the majority (63%) of whom were HPV16 positive. High
p16INK4A expression was significantly correlated with low histopathological grade (P ¼ 0.004), HPV positivity (P ¼ 0.032), and
long-term survival (P ¼ 0.045). High Ki-67 expression was negatively correlated with histopathological grade (Po0.001) and tumour
size (P ¼ 0.047). There was an association between HPV positivity and low histopathological grade, but not between HPV positivity
and survival.

Conclusion: High p16INK4A expression was associated with long-term survival, but the only independent predictors for survival
were tumour size and histopathological grade. Our results indicate that p16INK4A and Ki-67 expression might be useful in tumour
grading, and that it might be possible to use p16INK4A expression as a marker for HPV positivity, but this has to be further
elucidated.

Primary carcinoma of the vagina (PCV) is a rare malignancy, The importance of human papillomavirus (HPV) and its
comprising only 1–2% of the malignancies of the female genital oncogenic potential in the female genital tract, as well as in the
tract (Beller et al, 2006). The most prevalent histological type of oropharynx, has received considerable attention in the last decades.
PCV is squamous cell carcinoma (SCC). Primary carcinoma of the In accordance with other SCCs of the genital tract, such as cervical,
vagina mostly affects women over 60 years of age and has a poor vulvar, and anogenital carcinoma, the majority of PCV is
prognosis (Beller et al, 2006; Hellman et al, 2006). Due to its associated with high-risk HPV types, with a reported range of
rarity, biological and prognostic factors of PCV have not been 51.4–81% (Insinga et al, 2008; De Vuyst et al, 2009; Smith et al,
frequently studied, in contrast to other malignancies of the female 2009; Fuste et al, 2010; Brunner et al, 2011; Alonso et al, 2012).
genital tract. Two ethiopathogenic pathways have been postulated for PCV: one

*Correspondence: Dr K Hellman; E-mail: kristina.hellman@ki.se

Received 2 October 2013; revised 13 December 2013; accepted 9 January 2014; published online 13 February 2014
& 2014 Cancer Research UK. All rights reserved 0007 – 0920/14

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BRITISH JOURNAL OF CANCER PVC survival and HPV, p16INK4A and Ki-67

related to high-risk HPV positive, and the other to high-risk HPV- PCV, and subsequently treated at Karolinska University Hospital
negative PCV (Koyamatsu et al, 2003; Hellman et al, 2004; Fuste between 1978 and 1995. To get two groups with a significant
et al, 2010; Alonso et al, 2012). Knowledge of HPV status is difference in survival, patients with PCV were divided into
important in PCV because of its suggested clinical and prognostic short-term survivors (dying within p2 years of diagnosis) and
significance (Brunner et al, 2011; Alonso et al, 2012), but HPV long-term survivors (surviving X8 years after diagnosis). Of the
status is also important in understanding the aetiology of PCV. 130 consecutive patients, 77 fell into one of the two defined groups.
When HPV integrates in the host cell genome in squamous Archived tumour biopsies from these 77 patients were examined at
cells, the result is overexpression of the viral oncogenic proteins E6 the Department of Pathology, Karolinska University Hospital.
and E7, which interact with a number of specific cellular proteins All tumour biopsies were fixed in buffered formaldehyde, paraffin-
to initiate neoplastic transformation. The E7 protein binds to the embedded and diagnosed on haematoxylin and eosin-stained tissue
tumour suppressor protein, retinoblastoma gene product (pRB), sections.
leading to upregulation of CDKs, degradation of pRB, and For the present study, four sections from each archived tumour
enhanced expression of p16INK4A. Thus, an oncogenic HPV biopsy were prepared and used for histological diagnosis and
infection results in an accumulation of p16INK4A in the cell’s immunohistochemistry (thickness: 4 mm). Sections for haema-
nucleus and cytoplasm (Klaes et al, 2001). p16INK4A expression toxylin and eosin staining were prepared before and after each
measured by immunohistochemistry may be useful in clinical section to confirm tumour representativity. Primary carcinoma of
application as an indicator of degraded pRB caused by over- the vagina diagnoses, as well as the representativity of the sections
expression of the E7 protein. Studies in other SCCs have shown used for immunohistochemical studies, were reviewed and
that p16INK4A expression measured by immunohistochemistry confirmed by two pathologists (C Silfversward and E Wilander)
correlates well with HPV positivity (Mellin Dahlstrand et al, 2005; at the Department of Pathology, Karolinska University Hospital,
Santos et al, 2006; Reimers et al, 2007; Fuste et al, 2010), making and the Departments of Genetics and Pathology, and Clinical
it a useful biomarker for HPV-related oncogenic activity and Pathology and Cytology, Rudbeck Laboratory, Uppsala University
malignant transformation in cervix, vulva, and vagina Hospital and Uppsala University, Uppsala. The histopathologist
(Klaes et al, 2001; O’neill and Mccluggage, 2006; Fuste et al, reviewed and confirmed the histopathological grade and the
2010). In the cervix p16INK4A has proven as useful in identifying International Federation of Gynecology and Obstetrics (FIGO)
dysplasia, and correlates with the severity of dysplasia (Norman stage before immunohistochemistry was performed. Histopatho-
et al, 2007). In the cervix and other sites, it has also been logical evaluation was done according to the World Health
demonstrated to be of prognostic significance (Masoudi et al, 2006; Organization classification 1975, no 13, and staging was performed
Reimers et al, 2007; Schwarz et al, 2012). according to the FIGO staging system (accepted for PCV in 1963),
Ki-67 is a proliferation antigen, which is expressed in the nuclei based on the original clinical records and new histopathological
of growing cells. It is used to assess the proliferation index of results.
a cell population and can be used for grading dysplasia in cervical Nine tumour biopsies were excluded due to poor material
biopsies (Baak and Kruse, 2005). MIB-1 staining is reduced in with necrosis. Thus, 68 patients with PCV, 39 short-term and 29
atrophy and increased in dysplasia (Mittal et al, 1999), making it long-term survivors, were included in the final analyses. Sixty-three
useful in histopathologically uncertain cases as it can help tumour biopsies were classified as SCC, two as adenocarcinoma,
distinguish between postmenopausal changes and dysplasia. It and three as small cell carcinoma (Table 1). The study protocol was
has been suggested that higher MIB-1 immunostaining intensity is accepted by the ethical committee of the Stockholm County
an indicator of increased proliferation activity, which is associated Council (Dnr 01-194). Patient permission for use of archived
with unfavourable clinical outcome, increased tumour size, and tumour biopsies was not required.
more advanced stage of cancer (Heatley 1998; Kruse et al, 2003). Fifty-one patients received a combination of external beam
In cervical adenocarcinoma, increased Ki-67 expression was seen irradiation and brachytherapy, eleven patients received only
in tumours of lower grade and tumours with a higher stage at brachytherapy, and six patients were treated with surgery and/or
diagnosis, and correlated with a worse prognosis (Muller et al, chemotherapy alone. The medium external dose was 40 Gy
2008). It was observed that Ki-67 expression had a similar (range 19–60 Gy) and the medium dose to the vaginal tumour
prognostic value in vulvar carcinoma (Hantschmann et al, 2000). was 57 Gy (range 14–100 Gy). Patients were followed up at
Increased Ki-67 expression in PCV has previously been demon- 4-month intervals for 2 years and at 6-month intervals thereafter
strated, but has not been associated with survival (Koyamatsu et al, for an additional 3 years. After 5 years, patients were referred to a
2003; Habermann et al, 2004; Hellman et al, 2013). general gynaecologist for annual follow-up. All patients included in
Many prognostic factors for PCV have been studied, including the study had a reliable documented follow-up, with a minimum of
tumour stage, tumour size, and women’s age (Habermann et al, 8 years for the long-term survivors (Table 2).
2004; Hellman et al, 2006; Brunner et al, 2011; Jang et al, 2012). Recurrence was defined as disease reappearance 3 months or
Human papillomavirus-positive PCV has a better prognosis than more after completion of primary treatment in a patient
HPV-negative PCV in early stages (Alonso et al, 2012). p16INK4A considered to be in complete remission. Evidence of disease o3
expression measured by immunohistochemistry may be used to months after completion of treatment was defined as persistent
determine the HPV status of PCV (Fuste et al, 2010; Alonso et al, disease (Table 2).
2012). Prognostic factors and biomarkers of PCV need to be
HPV status. Briefly, analyses were performed on extracted
further elucidated to improve diagnostic tools and treatment. The
DNA obtained from a 10-mm thick section of paraffin blocks,
aim of this study is to determine HPV status and to investigate
the preceding section of which had been used for morphological
p16INK4A and Ki-67 expression and their correlation with clinical
diagnosis. These sections of archived tumour biopsies were
parameters and survival in patients with PCV.
dewaxed with xylene-ethanol. DNA was extracted by a MagNA
Pure LC Robot (Roche Diagnostics, Basel, Switzerland) according
to the manufacturer’s instructions.
MATERIALS AND METHODS HPV detection and typing. The quality of DNA samples was
analysed using a b-globin real-time PCR using 1 ml of the sample.
Patients and tumour biopsies. This study is based on a All samples that we included for future analysis were b-globin
population of 130 consecutive patients who were diagnosed with positive. Human papillomavirus testing was performed by PCR

1562 www.bjcancer.com | DOI:10.1038/bjc.2014.32

PVC survival and HPV, p16INK4A and Ki-67 BRITISH JOURNAL OF CANCER

Table 1. Patient and tumour characteristics Table 2. Follow-up data for 68 patients with PCV

Patients included in the study 68 Relapse/persistent disease

Age at diagnosis (years) Yes 40

No 28
Median 69
Mean 68 Follow-up time (days)

History of CIN Median 89

Mean 2808
Yes 21
No 47 Short- vs long-term survival

Previous gynaecological malignancy p2 years 39

X8 years 29
Yes 11
No 57 Vital status at last follow-up

Hysterectomy before diagnosis Alive 8

Dead of PCV 38
Yes 24
Dead of other disease 21
No 44
Alive with relapse 1
Histology Disease specific survival
Squamous cell carcinoma 63
Dead of PCV 38
Adenocarcinoma 2
All other 30
Small cell carcinoma 3
Overall survival
Histopathological grade (n ¼ 67)
Dead 59
Well differentiated 10
Alive 9
Moderately differentiated 34
Poorly differentiated 23 Abbreviation: PCV ¼ primary carcinoma of the vagina.

FIGO stage

I 34 et al, 2009). The Bioplex 200 Luminex system (Bio-Rad, Hercules,

II 13 CA, USA) was used for HPV detection and genotyping using
III 11 multiplex bead-based hybridisation with Luminex technology as
IV 10 described by Schmitt et al (2006). Briefly, 10 ml of the biotinylated
MGP-PCR product was mixed with beads coupled with different
Tumour size HPV probes. After 10 min of denaturation at 95 1C, the samples
o4 cm 29
were hybridised at 41 1C for 30 min. After washing, streptavidin-R-
4–8 cm 27 phycoerythrin was incubated with the samples for 30 min at room
48 cm 12 temperature. One hundred beads of each HPV type from each
sample were analysed using the Luminex system. Probes for 14
Tumour localisation oncogenic, high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52,
Upper third 36
56, 58, 59, 66, 68a, and 68b, including probes for variant sequences
Lower third 15
of HPV18, 35, 51, and 58) and 22 non-oncogenic types including
All other locations 17
low-risk and possible high-risk types (6, 11, 26, 30, 40, 42, 43, 53,
54, 61, 67, 70, 73, 74, 81, 82, 83, 86, 87, 89, 90, and 91) were used.
Growth pattern (n ¼ 59) Eleven negative controls (H2O) and eight positive controls (HPV
plasmid pools) were included in each test.
Exophytic 20
Ulcerating 31
Endophytic 8
Immunohistochemistry for p16INK4A and MIB-1. Immuno-
histochemical staining was performed with the CINtec Histology
Regional metastasis (inguinal node metastasis) Kit (Code No. K5336; DAKO Cytomation, Glostrup, Denmark)
according to the manufacturer’s recommendations using the Dako
Yes 4
Autostainer. This kit contains Tris EDTA buffer (  10) pH 9,
No 64
intended for epitope retrieval. A Coplin jar filled with epitope
Distant metastasis retrieval buffer, diluted 1 : 10 with distilled water, was placed in a
water bath and heated to 95–99 1C. Deparaffinised sections were
Yes 5 then incubated for 10 min while maintaining a temperature of
No 63 95–99 1C. Jars with slides were removed from the water bath and
left to cool at room temperature for 20 min, followed by washing
Abbreviations: CIN ¼ cervical intraepithelial neoplasia; FIGO ¼ International Federation of
Gynecology and Obstetrics. for 5 min in Wash Buffer (Code No. S3006; DAKO Cytomation)
diluted 1 : 10 with distilled water. The automated procedure began
with 1  rinse (1  rinse equals 4 min) in wash buffer diluted
amplification of a fragment in the L1 gene. Samples were tested for 1 : 10 with distilled water (Wash Buffer (  10), Code No. S3006;
the presence of HPV DNA by amplifying 1 ml of DNA with the DAKO Cytomation). Endogenous peroxidase activity was abol-
MGP primer system as previously described (Soderlund-Strand ished by incubating slides for 5 min in a peroxidase-blocking

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BRITISH JOURNAL OF CANCER PVC survival and HPV, p16INK4A and Ki-67

solution (Dako REAL, Code No. S2023; DAKO Cytomation). (10.3%) had no p16INK4A expression, eight (11.7%) had weak
Slides were rinsed 1  , after which 200 ml of the primary antibody expression, sixteen (23.5%) had moderate expression, and thirty-
against the p16INK4a protein clone E6H4 was dropped onto each seven (54.4%) had strong expression. A significant correlation was
slide, followed by incubation for 30 min. Slides were rinsed 1  . detected between p16INK4A expression and histopathological grade,
Reaction products were visualised by incubating slides for 30 min HPV status, and survival (Table 3). Increasing p16INK4A expression
with a visualisation reagent (a horseradish peroxidase/goat- correlated significantly with moderately and poorly differentiated
anti-mouse immunoglobulin-labelled dextran polymer) and, after tumours (P ¼ 0.004). Furthermore, higher p16INK4A expression
2  rinses, incubated for 10 min in a 1 : 40 solution of DAB correlated significantly with long-term survival (P ¼ 0.045,
chromogen (3.30 -diaminobenzidine) in DAB buffered substrate, Spearman correlation  0.24). Patients with moderate/high
also from the CINtec Histology Kit. Slides were then washed in expression of p16INK4A had better survival if the tumours were
distilled water for 1 min and counterstained for 2 min in Harris HPV negative compared with HPV-positive tumours (P ¼ 0.028),
Hematoxylin solution diluted 1 : 2 with distilled water. After 2 min data not shown.
of washing in water, slides were dehydrated in ethanol to xylene All patients with regional or distant metastasis had moderate or
and mounted in a water-free permanent mounting medium with strong p16INK4A expression; however, this was not significant due
mounting glass. Tissue sections containing cervical cancer were
used as positive controls for p16INK4a, while negative controls
Table 3. p16INK4A expression in correlation with clinical parameters and
consisted of incubated doublet slides in the negative control HPV status in 68 patients with PCV
reagent contained in the kit, instead of primary antibodies.
Immunostaining was independently evaluated by two observers
(C Flores-Staino and E Wilander) and was considered as positive Expression of p16
for p16INK4a when both observers agreed that the nuclei were
clearly stained. In addition, cells with a distinct cytoplasmic None/weak Moderate/strong
immunoreaction were scored as positive. Image analysis was Clinical parameters N (%) N (%) P-value
carried out as previously described. Immunohistochemistry results Age at diagnosis, year 75 (60–84) 66 (32–89) 0.084
were scored based on both staining intensity and percentage of (range)
immunoreactive epithelial cells (Sano et al, 1998; Klussmann et al, Histopathological grade (n ¼ 67)
2003). Scoring criteria for p16INK4a were no expression (negative);
weak expression (o30% positive cells); moderate expression Well 6 (60) 4 (40) 0.004
(31–50% positive cells); and strong expression (450% positive Moderate 5 (15) 29 (85)
cells). Samples scored as moderate or higher were considered as Poor 3 (13) 20 (87)
positive for p16INK4a (Sano et al, 1998; Klussmann et al, 2003).
Tumour size
To detect the Ki-67 antigen, we used the monoclonal mouse
antibody (clone MIB-1) (Code No. M7240; DAKO Cytomation). o4 cm 5 (17) 24 (83) 0.20
The Ki-67 antigen is a marker for mitotically active cells and is 4–8 cm 5 (19) 22 (81)
expressed in the nuclei of growing cells. Tumour biopsy sections 48 cm 5 (42) 7 (58)
were deparaffinised, rehydrated, and microwave treated in
FIGO stage
target retrieval solution diluted 1 : 10 with distilled water (Dako
REAL Target Retrieval Solution (  10) Code No. S2023; I–II 13 (28) 34 (72) 0.12
DAKO Cytomation) for 2  5 min at 500 W. Thereafter, slides III–IV 2 (10) 19 (90)
were subjected to the autostainer procedure and treated together
with the p16INK4a slides as described above. Ki-67 positivity was Local metastasis
scored with respect to nuclear staining and with attention Yes 3 (50) 3 (50) 0.12
to heterogeneity in distribution as follows: o10% positive cells; No 12 (19) 50 (81)
10–50% positive cells; and 450% positive cells.
Regional metastasis
Statistical analysis. Associations between ordinal variables were
Yes 0 (0) 4 (100) 0.27
tested using the Chi-square test or the Fisher’s exact test. When
No 15 (23) 49 (77)
investigating possible correlations between immunohistochemical
staining and clinical parameters, the semi-quantitative groups Distant metastasis
described above were analysed with Spearman’s rank correlation
coefficients. Yes 0 (0) 5 (100) 0.28
In the multivariate analyses, the median value was used to No 15 (24) 48 (76)
generate two groups of equal size for the immunohistochemical HPV status (n ¼ 44)
staining of Ki-67, with a cutoff at 50% positive cells. All parameters
that showed a significant difference when comparing short- and Positive 1 (5) 18 (95) 0.032
long-term survivors were then included in a multivariate logistic Negative 8 (32) 17 (68)
regression model to evaluate the independence of each factor.
Short- vs long-term survival
The median age in different groups was compared using the
Mann–Whitney test. p2 years 12 (31) 27 (69) 0.045
The covariates used in the statistical analysis are described in X8 years 3 (10) 26 (90)
Table 1.
Relapse

Yes 5 (28) 13 (72) 0.50

RESULTS No 10 (20) 40 (80)

Abbreviations: FIGO ¼ International Federation of Gynecology and Obstetrics;

Immunohistochemical expression of p16INK4A in relation to HPV ¼ human papillomavirus; PCV ¼ primary carcinoma of the vagina.
clinical parameters and HPV status. Seven patients with PCV

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PVC survival and HPV, p16INK4A and Ki-67 BRITISH JOURNAL OF CANCER

to the small number of patients that had metastasis. Patients (1 patient, 5.3%), HPV35 (1 patient), HPV56 (1 patient),
with strong p16INK4A expression were younger than those and HPV68 (1 patient). Human papillomavirus positivity was
with weak p16INK4A expression, although this difference was not significantly correlated with strong p16INK4A expression
significant. There was no correlation between relapse and the (P ¼ 0.032). One HPV16-positive patient was negative for
expression of p16. p16INK4A expression. All the other HPV-positive patients with
PCV (18 out of 19, 95%) showed either moderate or strong
Immunohistochemical expression of Ki-67 in relation to clinical
p16INK4A expression. The majority of HPV16-positive patients
parameters. Seven patients with PCV (10.3%) had o10% Ki-67-
with PCV showed strong p16INK4A expression, while those infected
positive cells, 38 (55.9%) had 10–50% positive cells, and 23 (33.8%)
with other HPV types showed either moderate or strong p16INK4A
had 450% positive cells. A significant correlation between Ki-67
expression. In all, 3 out of the 25 HPV-negative patients (12%)
expression and histopathological grade (Po0.001) and tumour size
were negative for p16INK4A immunostaining, while the remaining
(P ¼ 0.047) was found (Table 4). However, there was no correlation
88% showed varying expression: 17 out of 25 (68%) showed
detected between Ki-67 expression and long- and short-term
moderate or strong p16INK4A expression.
survival or relapse.
All HPV-positive patients with PCV showed moderate to strong
HPV types and status in correlation with clinical parameters proliferation activity; however, there was no significant correlation
and expression of p16INK4A and Ki-67. Forty-four out of between Ki-67 expression and HPV status (Table 4).
sixty-eight patients with PCV could be evaluated for HPV status. The mean age of HPV-positive and HPV-negative patients was
Nineteen (43%) were positive for high-risk HPV and twenty-five 72 years (range 51–89) and 68 years (range 32–84), respectively
(57%) were HPV negative (Table 5). The majority (12 out of 19, (Table 6). Hysterectomy was reported by 8 out of 19 HPV-positive
63%) of HPV-positive patients were positive for HPV16. The patients (42%), and 9 out of 25 (36%) HPV-negative patients.
others were positive for HPV45 (3 patients, 16%), HPV18 There was no difference in HPV positivity among women with a
history of CIN (seven HPV positive and nine HPV negative) or
hysterectomy (eight HPV positive and nine HPV negative). Seven
HPV-positive patients (7 out of 19, 37%) underwent hysterectomy
Table 4. Ki-67 expression in correlation with clinical parameters and HPV
status in 68 patients with PCV due to CIN 0–6 years before PCV diagnosis. Five HPV-negative
patients with PCV (5 out of 25, 20%) underwent hysterectomy due
to CIN 1–9 years before PCV diagnosis.
Ki-67 expression All of the HPV-positive patients with PCV had moderate to
poor histopathological grade (P ¼ 0.011). The two adenocarcino-
o10% 10–50% 450% mas were negative for HPV and had low expression of p16. Only
Clinical parameters P-value
N (%) N (%) N (%) one of the three small cell carcinomas was evaluable for HPV and
Histopathological grade (n ¼ 67) o0.001 that case was negative with moderate expression of p16. The other
Well 5 (50) 4 (40) 1 (10) two small cell carcinomas showed high expression of p16. Human
Moderate 1 (3) 23 (68) 10 (29) papillomavirus-positive patients were diagnosed at more advanced
Poor 1 (4) 10 (43) 12 (52) stages and the tumour was more commonly localised in the upper
Tumour size 0.047 third of the vagina. The growth pattern was more often ulcerative
in HPV-positive patients and exophytic in HPV-negative patients,
o4 cm 3 (10) 16 (55) 10 (35)
but this difference was not statistically significant. No significant
4–8 cm 3 (11) 11 (41) 13 (48)
48 cm 1 (8) 11 (92) 0 (0)
difference was seen in survival or relapse by HPV status.
FIGO stage 0.096 Survival analyses. Reliable follow-up data were available for all 68
I–II 7 (15) 23 (49) 17 (36) patients with PCV. In the univariate analysis, there was a
III–IV 0 (0) 15 (71) 6 (29) significant correlation between higher p16INK4A expression and
Local metastasis 0.056
long-term survival (Table 4). Larger tumour size and higher
FIGO stage correlated negatively with survival (Table 7). Locally
Yes 2 (33) 4 (67) 0 (0) advanced tumours with growth in the septum rectovaginale
No 5 (8) 34 (55) 23 (37)
correlated significantly with short-term survival (P ¼ 0.030). In
Regional metastasis 0.67 the multivariate analysis, the presence of local metastasis could not
Yes 0 (0) 2 (50) 2 (50) be included since there were no long-term survivors with local
No 7 (11) 36 (56) 21 (33)
Distant metastasis 0.73
Yes 0 (0) 3 (60) 2 (40) Table 5. p16INK4A expression in relation to HPV status and different HPV
No 7 (10) 35 (56) 21 (33) types

HPV status (n ¼ 44) 0.18

Positive 0 (0) 12 (63) 7 (37) HPV positive N (%)
Negative 4 (16) 14 (56) 7 (28) INK4A
p16 HPV negative Other HPV types
Short- vs long-term survival 0.46
HPV16
expression N (%) (18, 35, 45, 56, 68)
p2 years 5 (13) 23 (59) 11 (28) None 3 (12) 1 (5.3)
X8 years 2 (7) 15 (52) 12 (41) Weak (430%) 5 (20)
Relapse 0.50 Moderate (30–50%) 7 (28) 3 (16) 3 (16)
Yes 2 (11) 8 (44) 8 (44) Strong (450%) 10 (40) 8 (42) 4 (21)
No 5 (10) 30 (60) 15 (30)
Total 25 (100) 19 (100)
Abbreviations: FIGO ¼ International Federation of Gynecology and Obstetrics;
HPV ¼ human papillomavirus; PCV ¼ primary carcinoma of the vagina. Abbreviation: HPV ¼ human papillomavirus.

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BRITISH JOURNAL OF CANCER PVC survival and HPV, p16INK4A and Ki-67

Table 6. HPV status in relation to patient and tumour characteristics Table 7. Correlation between clinical characteristics, human
(n ¼ 44) papillomavirus (HPV), and survival

HPV HPV Survival

Patient and tumour positive negative
characteristics N (%) N (%) P-value Short term, p2 Long term, X8
Mean age at diagnosis 73 68 range years N (%) years N (%) P-value
(years) (range 51–89) (32–84)
Histopathological grade
History of CIN
Well 7 (70) 3 (30) 0.072
Yes 7 (44) 9 (56) 1.00 Moderate 23 (68) 11 (32)
No 12 (43) 16 (57) Poor 9 (39) 14 (61)
Previous gynaecological malignancy
Tumour size
Yes 3 (50) 3 (50) 1.00
o4 cm 5 (17) 24 (83) o0.001
No 16 (42) 22 (58)
4–8 cm 22 (81) 5 (19)
Hysterectomy before diagnosis 48 cm 12 (100) 0 (0)

Yes 8 (47) 9 (53) 0.76 FIGO stage

No 11 (41) 16 (59)
I–II 23 (49) 24 (51) 0.036
Histopathological grade (n ¼ 43) III–IV 16 (76) 5 (24)

Well 0 (0) 8 (100) 0.011 Local metastasis (tumour growth in septum rectovaginale)
Moderate 13 (62) 8 (38)
Poor 6 (43) 8 (57) Yes 6 (100) 0 (0) 0.030
No 33 (53) 29 (47)
FIGO stage
Regional metastasis (inguinal node metastasis)
I þ II 11 (34) 21 (66) 0.054
III þ IV 8 (67) 4 (33) Yes 2 (50) 2 (50) 1.00
No 37 (58) 27 (42)
Tumour size
o4 cm 6 (32) 13 (68) 0.40 Distant metastasis
4–8 cm 9 (53) 8 (47)
Yes 3 (60) 2 (40) 1.00
X8 cm 4 (50) 4 (50)
No 36 (57) 27 (43)
Tumour localisation
HPV status
Upper third 13 (54) 11 (46) 0.14
Lower third 2 (18) 9 (82) Positive 13 (68) 6 (32) 0.18
All other locations 4 (44) 5 (56) Negative 12 (48) 13 (52)

Growth pattern (n ¼ 39) Abbreviation: FIGO ¼ International Federation of Gynecology and Obstetrics.

Exophytic 5 (31) 11 (69) 0.11

Ulcerating 12 (63) 7 (37)
Endophytic 1 (25) 3 (75)
metastasis. The FIGO stage was also excluded from the analysis
Local metastasis since it did not add any additional influence after tumour size was
Yes 2 (50) 2 (50) 1.00 included. Thus, the parameters included in the analysis were histo-
No 17 (43) 23 (57) pathological grade, tumour size, Ki-67 expression, and p16INK4A
expression. Small tumour size and low histopathological grade
Regional metastasis
remained statistically significantly associated with long-term survival,
Yes 1 (50) 1 (50) 1.00 whereas Ki-67 expression and p16INK4A expression did not.
No 18 (43) 24 (57)

Distant metastasis DISCUSSION

Yes 1 (50) 1 (50) 1.00
No 18 (43) 24 (57) In this study, HPV status and the expression of molecular markers
p16INK4A and Ki-67 were evaluated as prognostic markers in 68
Short- vs long-term survival
patients diagnosed with PCV. There are some limitations to our
p2 years 13 (52) 12 (48) 0.18 study that should be addressed. This study was retrospective and
X8 years 6 (32) 13 (68) relied on archived tumour biopsies that were 410 years old.
Relapse
On the other hand, the study was based on one of the largest
collections of biological material in the literature to-date on HPV
Yes 3 (25) 9 (75) 0.18 and biological markers for this rare disease. Other studies
No 16 (50) 16 (50) addressing the prognostic value of HPV in PCV have included
Abbreviations: CIN ¼ cervical intraepithelial neoplasia; FIGO ¼ International Federation of
35–69 patients (Brunner et al, 2011; Alonso et al, 2012; Larsson
Gynecology and Obstetrics; HPV ¼ human papillomavirus. et al 2013), thus our study are among the larger ones.
Ki-67 expression correlated with histopathological grade and
tumour size, and p16INK4A expression correlated with

1566 www.bjcancer.com | DOI:10.1038/bjc.2014.32

PVC survival and HPV, p16INK4A and Ki-67 BRITISH JOURNAL OF CANCER

histopathological grade, HPV status, and survival. Interestingly, It has been demonstrated that oropharyngeal tumours with
both moderate and strong p16INK4A expression correlated with strong p16INK4A expression have a more favourable prognosis
better survival, which has not been demonstrated previously. As in regardless of HPV status, which is why the authors suggested that
other HPV-related cancer sites, there was a correlation between p16INK4A immunohistochemistry alone is the best test to use for
strong p16INK4A expression and the presence of HPV DNA. It has risk stratification and for predicting response to radiotherapy in
previously been reported that HPV is a positive prognostic factor this type of cancer (Lewis et al, 2010). p16INK4A expression has also
in PCV (Brunner et al, 2011; Alonso et al, 2012; Larsson et al, been shown to predict improved survival after chemoradiation
2013); p16INK4A expression was investigated in one of these studies therapy for advanced-stage invasive cervical carcinoma (Schwarz
(Alonso et al, 2012) and was not a marker for survival. Thus, the et al, 2012). p16INK4A is strongly expressed in HPV-related vulvar
role of p16INK4A expression as a marker for survival in PCV is still intraepithelial neoplasia but p16INK4A-negative vulvar intraepithe-
unclear. The inconsistent results might be explained by the limited lial neoplasia is not associated with HPV infection. Similarly, HPV-
number of patients included in both studies, and also by the positive invasive vulvar SCCs are p16INK4A positive, whereas the
different methods used to evaluate p16INK4A expression. In both more common non-HPV-related neoplasms are largely negative,
studies, there was a clear correlation between p16INK4A expression or focally positive (O’neill and Mccluggage, 2006). However,
and HPV status. In our study, HPV status could only be evaluated the prognostic and predictive value of p16INK4A expression has
in 65% of the patients, which could be one reason why we found no never been investigated in vulvar carcinoma. In the present study,
prognostic value. Likewise, Brunner et al (2011) found that we found that p16INK4A expression correlated with survival and
prognosis did not significantly differ between HPV-positive and also with HPV positivity. However, we observed that 17 out of 25
HPV-negative tumours in the entire cohort; however, patients with of the HPV-negative PCVs were p16INK4A positive and these also
unfavourable tumour stage and HPV positivity had improved had better prognosis than the p16-positive/HPV-positive tumours.
disease-free and overall survival. In multivariate analysis, Alonso This fact that many of the tumours showed moderate/high
et al (2012) confirmed better disease-free and overall survival of expression of p16INK4A also were HPV negative might indicate
HPV-positive patients independent of age and stage. This reduced that HPV-independent mechanisms also lead to overexpression
risk of progression and mortality in HPV-positive patients was of p16 in PCV, unless they are due to an undetected past or
limited to patients with stage I and II tumours. present infection. The p16 expression in HPV-negative tumours
Human papillomavirus positivity was detected in 43% of the needs to be further investigated to get increased knowledge in the
patients with PCV in our study, which is slightly lower compared aetiology of PCV.
with previously reported data from meta-analyses (Smith et al, The prognostic value of Ki-67 expression has been evaluated
2009) and other studies (Ferreira et al, 2008; Insinga et al, 2008; previously in PCV, but none was revealed (Hellman et al, 2013).
De Vuyst et al, 2009; Fuste et al, 2010; Brunner et al, 2011; Alonso In this study, other cutoff values were used to investigate Ki-67
et al, 2012; Larsson et al, 2013), with a prevalence ranging between expression more in detail. As in the earlier studies (Habermann
51.4% and 81%. This variation is most likely due to differences in et al, 2004; Hellman et al, 2013), we observed high proliferative
the detection methods used and in the selection of patients, but the activity in nearly all patients with PCV, and a correlation
geographical variation in HPV prevalence is another possibility. with tumour size was observed. However, no prognostic value
In the present study, HPV16 was the most prevalent type, as it was was found in the present study, leading us to conclude that Ki-67
found in 63% of patients, which is in accordance with previous case expression might not contribute any prognostic information to
series of patients with PCV (Fuste et al, 2010; Alonso et al, 2012; cases of PCV. On the other hand, our results indicate that Ki-67
Larsson et al, 2013), as well as patients with cervical and vulvar expression and p16INK4A expression might be useful in the
cancer. Other HPV types found were HPV18, 35, 45, 56, and 68, histopathological grading of PCV, that is, high Ki-67 and p16INK4A
which are all considered as high-risk types, and are also expression would indicate a low-grade tumour. Similar results have
occasionally found in other HPV-related cancers. been reported for cervical adenocarcinoma, where Ki-67 and
Studies on the spectrum of HPV types present in PCV are of p16INK4A expression may be a helpful marker in histopathological
particular importance with regard to the introduction of HPV grading (Muller et al, 2008).
vaccines, to evaluate their future effect on different diseases, Furthermore, Ki-67 and/or p16INK4A expression have been
including PCV. Larsson et al (2013) showed that HPV16-positive found to be markers of progression for intraepithelial neoplastic
patients with PCV had better survival than those infected with lesions in cervix, vulva, vagina, and anus (Cameron et al, 2002;
other HPV types. In a study of patients with cervical carcinoma Norman et al, 2007). We observed a correlation between HPV
treated with radiotherapy, patients infected with HPV16, or other positivity, lower histopathological grade, and high p16INK4A
types from the alpha-9 species, had a more favourable prognosis expression. In addition, p16INK4A expression correlated signifi-
(Wang et al, 2010; Lai et al, 2013). cantly with low histopathological grade. Likewise, Ki-67 expression
HPV16 is also the predominant type in oropharyngeal cancer. was associated with low histopathological grade. Taken together,
Human papillomavirus positivity has been shown to be a strong these findings might indicate that HPV-positive tumours have high
and independent prognostic factor of survival among patients with proliferative activity, and therefore respond better to radiation
oropharyngeal cancer, and has been related to better response to treatment, leading to a better prognosis. We also observed that
chemoradiation (Lindquist et al, 2007; Ang et al, 2010; Marur et al, smaller tumours had higher proliferative activity (higher Ki-67
2010; Gillison et al, 2012). This could be attributed to different expression) than larger tumours, which might also lead to a better
pathways of p53 dysfunction in HPV-positive and HPV-negative response to radiation therapy. On the other hand, different
tumours (Crook et al, 1991), but also to other cell cycle-related proliferative activity may exist in different parts of the tumour, and
changes that could affect radiosensitivity (Pyeon et al, 2007). a small biopsy only reflects the proliferative activity in that specific
Therefore, determination of HPV status is now part of routine part of the tumour. However, small tumours are more homo-
diagnostic evaluation when assigning prognoses for these malig- genous than large tumours, which have more genetic alterations.
nancies (Gillison et al, 2012). These alterations lead to more complex cellular dysfunctions
HPV18 is associated with less apoptosis than HPV16, which (Heselmeyer et al, 1997), resulting in increased resistance to
might result in increased radioresistance in HPV18-positive therapy and a worse prognosis.
cervical tumours. A possible mechanism could be a difference Human papillomavirus-positive tumours are known to be
in E6 oncoprotein activity (Arends et al, 1995; Hampson associated with less genetically complex tumours that respond
et al, 2001). better to therapy and have improved outcomes (Schwarz et al,

www.bjcancer.com | DOI:10.1038/bjc.2014.32 1567

BRITISH JOURNAL OF CANCER PVC survival and HPV, p16INK4A and Ki-67

2012). In the present study, almost all patients with PCV received since HPV18 is more prevalent in the cervix, where a glandular
radiation treatment, which is why it was difficult to compare transitional zone is present.
the response to radiation treatment between HPV-positive Obviously, other pathogenetic mechanisms are operating in the
and -negative patients in this study. But with regard to the development of PCV. As PCV predominantly occurs in post-
findings in oropharyngeal, cervical, and recent vaginal cancer menopausal women, which is in direct contrast to cervical
studies, the possibility to select patients for less aggressive therapies carcinoma where the majority of cases occur before 60 years of
based on HPV status and p16INK4A expression should be evaluated age, the response of these organs to the same carcinogenic stimulus
in larger studies. (e.g., HPV) seems to vary with age, and the response rate of the
In previous studies, we have found different clinical features and cervix may be more rapid (probably due to the transformation
expressions of molecular markers according to tumour location in zone) than the response rate of the vagina. There could also be an
the vagina (Hellman et al, 2004, 2006, 2013). Tumours located in additional factor that occurs predominantly in the older age group,
the upper third of the vagina have a better prognosis, and are often for example oestrogen deficiency, which makes the vaginal mucosa
associated with a history of CIN and younger age at diagnosis more responsive to carcinogenic stimuli, be it HPV or other
(Hellman et al, 2004, 2006). These findings have been correlated carcinogenic agents. However, what these other carcinogenic
with HPV positivity (Fuste et al, 2010; Alonso et al, 2012; Larsson agents might be is still relatively unknown. Irrespective of this,
et al, 2013). Fuste et al (2010) demonstrated that all tumours in the the vaginal squamous mucosa appears to be relatively resistant to
upper third of the vagina were HPV positive (none of the HPV- malignant transformation in comparison with the squamous
negative tumours were located in the upper third of the vagina), mucosa of other topographic sites.
and that HPV-positive tumours tend to affect younger women and In summary, there is now accumulating evidence that there are
women with a history of CIN. Alonso et al (2012) and Larsson et al two types of PCV, like vulvar carcinoma, which seem to develop
(2013) found superior survival among patients with HPV-positive along different pathogenetic pathways and have different risk
tumours. In the present study, HPV-positive tumours were more factor profiles: one subset that is HPV related, and one that is not.
often located in the upper third of the vagina than HPV-negative Studies have shown that HPV-positive tumours have a better
tumours. However, we found no difference in age at diagnosis prognosis, are diagnosed at a younger age, are associated with a
or history of CIN between HPV-positive and HPV-negative history of CIN, and are often located in the upper third of the
patients. vagina. Histopathologically, HPV-positive tumours are mostly
In a case series of advanced cervical carcinoma, Schwarz et al non-keratinising, and have strong p16INK4A expression. In
(2012) found an association between p16INK4A-negative patients contrast, HPV-negative PCV is often of the keratinising type,
with SCC increased age at presentation, and suggested that and occurs in older patients.
p16INK4A expression might be an age-related factor. This finding In conclusion, we have for the first time found that p16INK4A
is consistent with the results from the head-and-neck literature, expression might be a prognostic marker in PCV. However, the
which document an increased incidence of HPV-related SCC with only factors we found that could independently predict survival
p16INK4A upregulation in younger patients with no other known were tumour size and histopathological grade. There was no
carcinogen exposure, whereas HPV-negative tumours are more difference in survival by HPV status in this study population, but
common in older patients with documented carcinogen exposure we found an association between HPV positivity and p16INK4A
(Marur et al, 2010; Gillison et al, 2012). In our study, we also found expression, and between p16INK4A expression, Ki-67 expression,
that older patients had lower p16INK4A expression than younger and histopathological grade that might be useful in tumour
patients. In addition, HPV-negative vaginal tumours are more grading. p16INK4A immunohistochemistry could be a marker for
often seen in older patients (Fuste et al, 2010; Alonso et al, 2012), diagnosis of HPV-positive PCV and also a prognostic marker for
but this could not be verified in our material. therapeutic guidance. Further studies are needed to confirm these
Furthermore, HPV-positive PCV is more frequently of non- findings, and to evaluate p16INK4A and HPV status as markers for
keratinising, basaloid, and warty type than HPV-negative diagnosis, prognosis, and therapy. Furthermore, studies on the
PCV (Fuste et al, 2010). In addition, keratinising PCV occurs aetiology of HPV-negative PCV are warranted.
more frequently in older patients, whereas non-keratinising
PCV more frequently affects younger women (Ferreira et al,
2008). It has been reported that keratinising PCV is less
radiosensitive, and is associated with shorter overall survival ACKNOWLEDGEMENTS
(Kumar et al, 2009). Clearly, several factors are associated with age,
and other pathogenetic mechanisms that may also influence
We thank Carmen Flores-Staino for technical assistance and Trudy
response to therapy and outcome seem to operate at older age.
Perdrix-Thoma for editing assistance and English language review.
Genital HPV infections are sexually transmitted and in HPV-
This study was supported by the Swedish Cancer Foundation
positive women the vaginal mucosa is exposed to higher
(070623, CAN 2007/1044, 11 0544, CAN 2011/471), Karolinska
concentrations of virus particles than the cervical canal
Institutet Cancer Strategic Grants (5888/05-722), the Swedish
(Gustavsson et al, 2009). Despite this, the oncogenic effects of
Research Council (521-2008-2899), the Medical Research Council,
HPV infection are more profound in the cervix compared with the
and the Cancer Society in Stockholm, the Stockholm County
vagina, since almost all cervical carcinomas are caused by HPV,
Council, Swedish Labour Market Insurance and by Lion’s Cancer
whereas HPV is only found in around 50% of patients with PCV.
Research Foundation, University of Umeå. We thank Associate
The reason for this discrepancy is probably the existence of the
Professor Claes Silfversward, Department of Pathology at
transitional zone between the stratified squamous epithelium and
Karolinska University Hospital for valuable help with re-evaluating
cervical glandular epithelium. In this area, special target cells have
histopathological diagnoses.
been identified that are highly susceptible to malignant transfor-
mation due to HPV infection (Herfs et al, 2012). The vaginal
mucosa, like vulvar mucosa and penis, lacks such vulnerable sites
and for this reason they are probably more resistant to HPV-
related SCC. In HPV-positive PCV, HPV16 seems to predominate CONFLICT OF INTEREST
to a higher degree than in cervical SCC. It is possible that the lack
of glandular epithelium in the vagina contributes to this picture, The authors declare no conflict of interest.

1568 www.bjcancer.com | DOI:10.1038/bjc.2014.32

PVC survival and HPV, p16INK4A and Ki-67 BRITISH JOURNAL OF CANCER

the age at diagnosis. The Radiumhemmet series 1956-96. Int J Gynecol

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1570 www.bjcancer.com | DOI:10.1038/bjc.2014.32