Neuro-Oncology

Neuro-Oncology 16(2), 298 –302, 2014

doi:10.1093/neuonc/not140
Advance Access date 26 November 2013

Evidence of association of human papillomavirus with prognosis

worsening in glioblastoma multiforme
Michele Vidone, Federica Alessandrini, Gianluca Marucci, Anna Farnedi, Dario de Biase, Fulvio Ricceri,
Claudia Calabrese, Ivana Kurelac, Anna Maria Porcelli, Monica Cricca, and Giuseppe Gasparre

Downloaded from https://academic.oup.com/neuro-oncology/article-abstract/16/2/298/1082242 by guest on 01 June 2020

Dip. Scienze Mediche e Chirurgiche, Unità di Genetica Medica, Università di Bologna, policlinico S.Orsola-Malpighi, Bologna, Italy (M.V., C.C.,
I.K., G.G.); Dip. Medicina Specialistica, Diagnostica e Sperimentale, Unità di Virologia, Università di Bologna, policlinico S.Orsola-Malpighi, via
Massarenti 9, Bologna, Italy (F.A., M.C.); Dip. Scienze Biomediche e Neuromotorie, Università di Bologna, Sezione di Patologia “M.Malpighi”
Ospedale Bellaria, Bologna, Italy (G.M., A.F.); Dip. Medicina Specialistica, Diagnostica e Sperimentale, Unità di Patologia, Università di Bologna,
Ospedale Bellaria, via Altura 3, Bologna, Italy (D.d.B.); AO Ordine Mauriziano di Torino, via Magellano 1, Torino, Italy (F.R.); Dip. Farmacia e
Biotecnologie, Università di Bologna, Bologna, Italy (A.M.P.); Centro Interdipartimentale di Ricerca Industriale, Scienze della Vita e Tecnologie
per la Salute, Università di Bologna, Bologna, Italy (A.M.P.)

Corresponding author: Giuseppe Gasparre, PhD, Dipartimento di Scienze Mediche e Chirurgiche, UO Genetica Medica, Pad.11, Policlinico S.Orsola-Malpighi,
via Massarenti 9, 40138, Bologna, Italy (giuseppe.gasparre@gmail.com).

Background: Glioblastoma multiforme (GBM) is the most malignant brain tumor in adults, but its etiology still remains unknown. Recently,
a role of viruses such as cytomegalovirus and JC virus in gliomagenesis has been suggested. Since human papillomavirus (HPV) is con-
sidered the most common oncogenic virus in humans, we evaluated its occurrence in GBM samples.
Material and Methods: Fifty-two formalin-fixed paraffin-embedded primary glioblastoma specimens were retrospectively analyzed. The
presence of HPV genome on tumor DNA was assessed by MY/GP nested PCR. Confirmation of HPV detection was obtained by chromogenic
in situ hybridization (CISH) and immunohistochemistry (IHC) with an antibody directed against the L1 capsidic protein. Finally, univariate
and multivariate proportional-hazards models were used to compare the risk of death among HPV-positive and HPV-negative patients.
Results: Strikingly, viral DNAwas detected after PCR in 12 cases (23%). HPV16 genome was present in 25% infected samples, whereas the
remaining samples tested positive for HPV6. CISH confirmed positivity in all infected samples for which enough material was available.
Moreover, IHC positivity suggested that production of viral proteins from HPV genome is an ongoing process in GBM cancer cells. Finally an
association between HPV infection and a worse prognosis was found in patients upon age stratification with a univariate analysis (HR,
2.10; 95% CI, 1.00–4.44; log-rank P ¼ .045).
Conclusions: HPV infection status may be considered an independent prognostic factor in GBM patients and suggests that prevention
may be considered, should HPV be recognized as a causative agent in gliomagenesis.

Keywords: glioblastoma multiforme, HPV, prognosis.

Glioblastoma multiforme (GBM) (WHO astrocytoma grade IV) is
the most common primary malignant brain cancer in adults1
and is associated with a median survival of 12 to15 months from
diagnosis.2 Standard treatments consist of total surgical resection
whenever possible, followed by a combination of the alkylating
cytostatic drug temozolomide (TMZ) and radiotherapy (RT),
which however provide relatively limited improvements.3 The eti-
ology of GBM is yet unknown, albeit recently a consensus
towards a possible association with cytomegalovirus (CMV) or
JCV has been proposed.4,5 We show here for the first time that
oncogenic human papillomavirus (HPV) is present at a relevant fre-
quency in GBM specimens and associates with a poorer prognosis.
To this aim, 52 primary GBM cases consecutively referred to the
Bellaria Hospital, Bologna, Italy, were retrieved (27 men and 25
women); their clinical and anagraphical features are summarized
in Table 1. Patients ranged in age from 41 to 78 years (mean ¼
62y) at diagnosis. Standard treatment regimen (TMZ concurrent
with or adjuvant to RT) was administered in 27 patients, whereas
2 patients were treated with RT alone, and 19 patients did not
receive any adjuvant therapy. Information about therapy was
not available in 7 cases.
In order to assess the presence of HPV in GBM specimens, DNA
extracted from tumor samples was analyzed by nested PCR with
the MY/GP primer sets.6 Surprisingly 12 of 52 (23%) cases were

Received 25 April 2013; accepted 29 July 2013

# The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com.

298
 Vidone et al.: HPV in prognosis of glioblastoma

Table 1. Clinical and anagraphical data of GBM patients

Case Sex Age Therapy MGMT Follow-up HPV CISH

Methylation (months) Genotype Positivity
CH RT Status

GBM1 M 53 unknown unknown np 0.2 HPV6 N/A

GBM2 F 65 N N N 17 -
GBM3 F 70 Y Y Y 7.3 -

Downloaded from https://academic.oup.com/neuro-oncology/article-abstract/16/2/298/1082242 by guest on 01 June 2020

GBM4 M 59 Y Y nd 28.7 -
GBM5 F 68 Y Y N 7.1 HPV16 +
GBM6 M 71 unknown unknown N 14 -
GBM7 F 65 N N N 6.9 -
GBM8 F 73 Y Y Y alive 38 -
GBM9 F 66 unknown unknown N 22 -
GBM10 F 72 N N Y 6 -
GBM11 M 78 N N N 1.8 -
GBM12 F 41 Y Y Y 23.1 -
GBM13 F 61 N Y N 3.9 -
GBM14 F 55 Y Y Y alive 34.7 -
GBM15 M 60 N N N 5.1 -
GBM16 M 77 N N Y 4.3 HPV16 +
GBM17 M 69 N N N 13.8 -
GBM18 F 75 N Y Y 8.8 -
GBM19 F 74 N N N 7.2 HPV16 +
GBM20 F 69 N N Y 2 HPV6 +
GBM21 M 57 Y Y Y 9 -
GBM22 M 70 unknown unknown N 6 HPV6 +
GBM23 M 45 Y Y N alive 27.3 HPV6 N/A
GBM24 M 72 Y Y N 16.8 HPV6 N/A
GBM25 F 75 Y N Y 15.8 HPV6 N/A
GBM26 M 61 unknown unknown N 7 HPV6 N/A
GBM27 M 76 unknown unknown Y 4 -
GBM28 M 50 Y Y N 19 -
GBM29 F 49 N N Y alive 13 -
GBM30 M 75 Y Y N alive 13.1 -
GBM31 F 47 N N Y 16.8 -
GBM32 F 68 N N N alive 18 -
GBM33 M 71 Y Y Y 3 -
GBM34 F 53 N N N 6 -
GBM35 M 59 Y Y nd 17 -
GBM36 F 65 Y Y nd 7 -
GBM37 F 50 Y N N 4.1 -
GBM38 F 68 Y Y Y 2.9 -
GBM39 F 59 Y Y Y 25.2 -
GBM40 M 58 Y Y N 29 -
GBM41 M 41 Y Y Y 16.3 HPV6 N/A
GBM42 M 51 Y Y nd 17 HPV6 N/A
GBM43 M 53 Y Y Y 2.4 -
GBM44 M 74 N N nd 9 -
GBM45 F 49 N N N 24 -
GBM46 M 53 Y Y nd 11 -
GBM47 M 63 Y Y N alive 23 -
GBM48 M 59 Y Y nd 16 -
GBM49 F 63 N N nd 5 -
GBM50 M 72 N N nd alive 18 -

Continued

Neuro-Oncology 299
Vidone et al.: HPV in prognosis of glioblastoma

Table 1. Continued

Case Sex Age Therapy MGMT Follow-up HPV CISH

Methylation (months) Genotype Positivity
CH RT Status

GBM51 M 60 Y Y Y 16 -
GBM52 F 72 N N Y alive 14.7 -

Downloaded from https://academic.oup.com/neuro-oncology/article-abstract/16/2/298/1082242 by guest on 01 June 2020

Abbreviations: CH, Chemotherapy; RT, radiotherapy; nd, not detected; N/A, tissue not available.
Age is intended at diagnosis. Follow-up column indicates the survival time (months) after surgery.

found to be positive for HPV amplification. Sequencing of PCR pro-
ducts and BLAST alignment were performed to identify the viral
genotype associated with the infection. Interestingly, 3 cases
showed 100% of homology with HPV16 genome, the prevalent
high-risk type in cervical cancer, whereas the remaining 9 cases
harbored HPV6, the most relevant low-risk type in benign anogeni-
tal lesions (Fig. 1A).
Confirmation of localization of HPV genome within cancer cells
was carried out by chromogenic in situ hybridization (CISH) on
infected and noninfected samples for which enough material was
available (Supplementary material, Material and Methods). CISH
ascertained positivity in all infected cases, 2 of which showed an
intense widespread nuclear positivity (Table 1, Fig. 1B). Surprisingly
in 1 sample, the signal was not restricted to the stromal cancer
cells but was also evident in endothelial cells. Such positivity may re-
present a seminal observation of either a route of entry into the brain
or viral spreading within the glial tissue, although functional confir-
mations are warranted. In this regard, the possibility of HPV diffusion
via the bloodstream has already been hypothesized, since the viral
genome has been detected in the blood of patients with cervical
and oropharyngeal HPV-associated cancers.7,8
To date, there is only 1 report on HPV occurrence in the brain in
association with cortical dysplasia.9 Another recent study reported
the presence of HPV in temporal arteritis;10 interestingly here both
high- and low-risk types were detected, indicating that the capabil-
ity to reach the cerebral districts is not restricted to high-risk HPVs.
To assess a functional effect of HPV genome occurrence in GBM
samples, we evaluated production of viral proteins by immunohis-
tochemistry (IHC) with an antibody directed against the capsidic
protein L1. Again, cases for which enough material was available
showed positivity for L1 staining, suggesting production of viral
proteins from HPV genome to be an ongoing process in GBM
cancer cells (Fig. 1C).
We next attempted to ascertain whether a correlation could be
found between occurrence of HPV infection and prognosis in GBM.
To date, few biomarkers have proven to be of prognostic significance
with respect to the survival of patients affected by GBM.11 It has
been reported that the methylation of O6-methylguanine-DNA
methyltransferase (MGMT) gene, which encodes a DNA repair
enzyme that catalyzes the transfer of mutagenic and cytotoxic
adducts from O6 guanine in DNA, is associated with improved sen-
sitivity to chemotherapeutics such as TMZ.12 MGMT methylation
status had been previously assessed in most of our cases, and its
promoter had been found to be methylated in 20 cases (Table 1).
The evaluation of the effect of MGMT methylation status on patients
survival did not result in any significant change in prognosis, either
alone or in association with TMZ treatment (univariate Cox model
HR ¼ 0.96; 95% CI. 0.49–1.90) (multivariate Cox model HR ¼ 0.80;
95% CI, 0.38–1.71).
Infection by HPV in cancers of different organs is often asso-
ciated with either a positive or negative prognostic effect whose
entity depends on the tissue and the viral type.13 – 17 Therefore,
we speculated that HPV may also have an impact in GBM. Kaplan –
Meier curves show that patients with HPV-positive GBM had a worse
median overall survival than those with HPV-negative cancer. In
fact, the 12-month rates of overall survival were 41% and 51%, re-
spectively. Nonetheless, there was no statistically significant differ-
ence in survival rate between the 2 groups, which was likely due to
the need for an enlargement of our cohort. A trend for a shorter
median overall survival was indeed observed in patients with
HPV-positive GBM (7.3 months vs. 11 months) (log-rank P ¼ .19)
(Fig. 1D).
It has been ascertained that 50 years is usually a more appropri-
ate and reproducible cut-off age for clinical subdivision of patients
with GBM into prognostically relevant subsets.18,19 In this group, a
statistically significant worsening of the prognosis was observed in
HPV-positive patients aged 50 years and older after univariate ana-
lysis (HR, 2.10; 95% CI, 1.00–4.44; log-rank P ¼ .045; median
overall survival ¼ 7.3 vs. 11; Fig. 1E), confirmed by the borderline
significance found in multivariate analysis (HR,2.48; 95% CI,
0.99 –6.24). No statistically significant differences were reported
for age, MGMT methylation status, and treatment between the 2
groups (Fig. 1F).
Overall, we show here for the first time that GBM may be asso-
ciated with HPV infection and that prognosis is poorer when such
infection occurs.
HPV is considered the causative agent for nearly all cases of cer-
vical cancer and has been associated with an increasing number of
other tumors such as ovarian, oropharyngeal, and lung cancers. Its
transforming capabilities are due to the expression of E6 and E7
proteins, which deregulate cell proliferation and apoptosis upon
binding to p53 and pRb.20 Although functional studies on the in-
volvement of HPV in the origin of GBM were beyond the scope of
this preliminary analysis, we speculate that the virus may be a con-
current cause of gliomagenesis, with additional genetic hits being
likely required for HPV-related tumorigenesis, such as the activa-
tion of oncogenes.
A relevant issue that deserves further investigation is the deter-
mination of the physical state in which HPV exists in a nonepithelial
cancer such as GBM. In invasive cervical neoplasms and advanced
precancerous lesions, HPV is generally found in an integrated, non-
replicating state characterized by the overexpression of E6 and E7
proteins. The production of viral particles occurs only in low-grade
precancerous lesions or nondysplastic tissues and requires a

300
 Vidone et al.: HPV in prognosis of glioblastoma

Downloaded from https://academic.oup.com/neuro-oncology/article-abstract/16/2/298/1082242 by guest on 01 June 2020

Fig. 1. (A) Pairwise alignment between the HPV L1 protein sequence and the query obtained translating the nucleotide sequence detected by PCR (see
methods). (B) Chromogenic in situ hybridization of a HPV-positive case (left panel) and a HPV-negative control (right panel); the arrow indicates the
nucleus of an endothelial cell showing positivity. (C) Immunohistochemistry staining for L1 capsidic protein. The left panel shows a positive case that
displays a classical nuclear positivity to L1 compared to the negative control (right panel). (D) Kaplan– Meier estimates of the survival among GBM
patients under study according to HPV status. Patients with HPV-positive tumors have a worse median overall survival than patients with HPV negative
tumors (median overall survival 7.3 vs. 11 months; log-rank test P ¼ .19). (E) Kaplan–Meier estimates of the survival among GBM patients according to
HPV status within the subset of patients older than 50 (log-rank P ¼ .045, median overall survival ¼ 7.3 vs. 11). (F) Summary of the clinical
characteristics of HPV-positive and HPV-negative patient subgroups. P values were calculated with t-test or Fisher’s exact test.

Neuro-Oncology 301
Vidone et al.: HPV in prognosis of glioblastoma
pluristratified epithelium.20 Thus, it is likely that in GBM the virus
may be associated with an abortive infection with transforming
properties, since viral proteins were detected here by IHC.
Although HPV is considered to be an exclusive epitheliotropic
pathogen, it has been demonstrated that virions are able to bind
to cells derived from several tissues and species.21 Moreover, the
heparan sulphate proteoglycans, which are likely to be the initial
binding molecules for many HPV types, are also found to be
expressed in astrocytes.22 Whether these molecules may mediate
access of HPV in GBM initiating cells warrants investigation.
It is worth mentioning that all of our patients had received
steroid treatment for a period ranging from 7 to 15 days before
surgery. Albeit prolonged steroid administration may contribute
to the reactivation of neurotropic viruses, dosage and duration of
the treatment here may not be sufficient to induce immunode-
pression and may hardly justify a de novo HPV infection that may
extend quickly from endothelia to most neoplastic cells, unless
the virus was already present in the glia in a quiescent state.
Lastly, it is significant that among few lower-grade astrocyto-
mas analyzed and subsequently excluded from the cohort for
the sake of cases homogeneity was 1 case out of 3 cases positive
to HPV6. Our findings may prompt further investigation into the po-
tential etiological or modifying role of HPV in gliomagenesis, since
preventive measures to avoid HPV-induced cervical cancer already
exist,23 and shed light on the search for molecular determinants of
this devastating neoplasia.
Supplementary Material
Supplementary material is available at Neuro-Oncology Journal
online (http://neuro-oncology.oxfordjournals.org/).
Funding
This work was supported by the Associazione Italiana Ricerca sul Cancro -
AIRC [IG8810]; by the Italian Ministry of University MIUR - Futuro in
Ricerca 2008 [J31J10000040001] and Fondazione Umberto Veronesi -
DISCO TRIP project to GG; MV was supported by a fellowship from the Asso-
ciazione Italiana Tumori Cerebrali - AITC; IK is supported by AIRC triennial
fellowship ‘Borromeo’.
Acknowledgments
We thank Mr. M. Gualandi and Mr. E. Iannetti for technical support.
Conflict of interest statement. None declared.
References
1. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med. 2008;
359(5):492– 507.
2. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus
concomitant and adjuvant temozolomide for glioblastoma. N Engl J
Med. 2005;352(10):987– 996.
3. Stupp R, Hegi ME, Mason WP, et al. Effects of radiotherapy with
concomitant and adjuvant temozolomide versus radiotherapy alone
on survival in glioblastoma in a randomised phase III study: 5-year
analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10(5):459– 466.
302
4. Dziurzynski K, Chang SM, Heimberger AB, et al. Consensus on the role of
human cytomegalovirus in glioblastoma. Neuro Oncol. 2012;14(3):
246–255.
5. Del Valle L, Gordon J, Assimakopoulou M, et al. Detection of JC virus DNA
sequences and expression of the viral regulatory protein T-antigen
in tumors of the central nervous system. Cancer Res. 2001;61(10):
4287–4293.
6. Gravitt PE, Peyton CL, Alessi T, et al. Improved amplification of genital
human papillomaviruses. J Clin Microbiol. 2000;38(1):357–361.
Downloaded from https://academic.oup.com/neuro-oncology/article-abstract/16/2/298/1082242 by guest on 01 June 2020
7. Capone RB, Pai SI, Koch WM, et al. Detection and quantitation of human
papilloma virus (HPV) in the sera of patients with HPV-associated
Head and Neck Squamous cell carcinoma. Clin Cancer Res. 2000;6(11):
4171–4175.
8. Bodaghi S, Wood LV, Roby G, et al. Could human papillomaviruses be
spread through blood? J Clin Microbiol. 2005;43(11):5428– 5434.
9. Chen J, Tsai V, Parker WE, Aronica E, Baybis M, Crino PB. Detection of
Human Papillomavirus in human focal cortical dysplasia Type IIB.
Ann Neurol. 2012;72(6):881 –892.
10. Mohammadi A, Pfeifer JD, Lewis JS Jr. Association between human
papillomavirus DNA and temporal arteritis. BMC Musculoskelet Disord.
2012;13:132.
11. Ducray F, Idbaih A, Wang XW, Cheneau C, Labussiere M, Sanson M.
Predictive and prognostic factors for gliomas. Expert Rev Anticancer
Ther. 2011;11(5):781– 789.
12. Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit
from temozolomide in glioblastoma. N Engl J Med. 2005;352(10):
997– 1003.
13. de Boer MA, Jordanova ES, Kenter GG, et al. High human papillomavirus
oncogene mRNA expression and not viral DNA load is associated with
poor prognosis in cervical cancer patients. Clin Cancer Res. 2007;13:
132– 138.
14. Lee LA, Huang CG, Liao CT , et al. Human papillomavirus-16 infection in
advanced oral cavity cancer patients is related to an increased risk of
distant metastases and poor survival. Plos One. 2012;7(7):e40767.
15. Rautava J, Kuuskoski J, Syrjänen K, Grenman R, Syrjänen S. HPV
genotypes and their prognostic significance in head and neck
squamous cell carcinomas. J Clin Virol. 2012;53(2):116–120.
16. Yhim HY, Lee NR, Song EK, et al. The prognostic significance of tumor
human papillomavirus status for patients with anal squamous cell
carcinoma treated with combined chemoradiotherapy. Int J Cancer.
2011;129(7):1752–1760.
17. Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human
papillomavirus-positive head and neck squamous cell carcinoma in a
prospective clinical trial. J Natl Cancer Inst. 2008;100(4):261–269.
18. Ohgaki H, Dessen P, Jourde B, et al. Genetic pathways to glioblastoma: A
population-based study. Cancer Res. 2004;64(19):6892–6899.
19. Korshuno A, Sycheva R, Golanov A. The prognostic relevance of
molecular alterations in glioblastomas for patients age ,50 years.
Cancer. 2005;104(4):825– 832.
20. Moody CA, Laimins LA. Human papillomavirus oncoproteins: pathways
to transformation. Nat Rev Cancer. 2010;10(8):550–560.
21. Muller M, Gissmann L, Cristiano RJ, et al. Papillomavirus capsid binding
and uptake by cells from different tissues and species. J Virol. 1995;
69(2):948–954.
22. Properzi F, Lin R, Kwok J, et al. Heparan sulphate proteoglycans in glia
and in the normal and injured CNS: expression of sulphotransferases
and changes in sulphation. Eur J Neurosci. 2008;27(3):593–604.
23. Campo MS, Roden RB. Papillomavirus prophylactic vaccines: established
successes, new approaches. J Virol. 2010;84(3):1214–1220.