RESEARCH ARTICLE
No Evidence for Human Papillomavirus Infection in Focal Cortical Dysplasia IIb Roland Coras, MD,1 Klaus Korn, MD,2 Christian G. Bien, MD,3 Thilo Kalbhenn, MD,4 Karl R€ ossler, MD,5 Katja Kobow, PhD,1 Johannes Giedl, MD,6 Bernhard Fleckenstein, MD, PhD,2 and Ingmar Blumcke, MD1 Objective: The etiology of focal cortical dysplasia type IIb (FCDIIb) remains enigmatic in patients suffering from drug-resistant epilepsy, and an aberrant activation of the mammalian target of rapamycin complex 1 signaling pathway (mTORC1) was detected in this developmental brain malformation. Recently, the human papillomavirus (HPV) oncoprotein E6 has been identified as a potent activator of mTORC1, and HPV16 E6 has been described to persist in balloon cells obtained from surgical FCDIIb specimens. Although this observation was replicated by an independent second report, it contradicts current knowledge of HPV biology. HPV infects the squamous or mucocutaneous epithelium; hematogenic spread into other tissues has not been observed. In addition, brain carcinogenesis has never been reported in FCDIIb patients. Herein, we have tried to confirm 2 previous reports of HPV16 E6 infection using an independent series of 14 surgical specimens with histopathologically confirmed FCDIIb. Methods: Snap-frozen FCDIIb specimens were tested for HPV DNA using the primer set for amplification of the complete E6 reading frame of HPV16 and 3 other sets of primers (2 consensus primer sets detecting multiple HPV genotypes, and another primer set specifically used for HPV16). Furthermore, formalin-fixed and paraffin-embedded histopathological preparations were immunohistochemically analyzed using previously described antibodies directed against the HPV E6 oncoprotein. Results: All 14 FCDIIb specimens were negative for HPV DNA with all 4 primer sets. Antibodies directed against the HPV E6 epitope showed weak labeling of cytoplasm in balloon cells, as previously described in FCDIIb, but also in other cell populations. Interpretation: Our data did not confirm previously reported evidence for HPV16 detection in FCDIIb. ANN NEUROL 2015;77:312–319
H
uman papillomaviruses (HPVs) belong to a DNA virus family capable of infecting keratinocytes of the skin or mucous membranes. Their causative role in cervical carcinomas was first postulated by Harald zur Hausen in 1976, and since then has been studied and confirmed in many human cancers, including squamous cell carcinomas of the head and neck.1,2 More than 150 different HPV types have been reported, and at least 15 types are considered as carcinogenic.3 Carcinogenesis is conveyed by E6 and E7 oncoproteins of high-risk HPV types, especially HPV16 and HPV18. E6 binds to p53,4 and E7 to the retinoblastoma tumor suppressor gene product
pRB,5 thereby effecting similar tumor transformation pathways. More recently, HPV16 E6 expression was shown to also cause an increase in mammalian target of rapamycin complex 1 signaling pathway (mTORC1) activity through an enhanced phosphorylation of mTOR and activation of downstream signaling pathways (ie, Akt and S6K).6 mTOR activation is considered an important molecular hallmark also in focal cortical dysplasia type IIb (FCDIIb), as shown either by genetic TSC1 analysis or immunohistochemistry for activated downstream targets.7–11 Considering the well-characterized biology and epidemiology of high-risk HPV genotypes, 2 recent
View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24328 Received Jul 9, 2014, and in revised form Nov 24, 2014. Accepted for publication Nov 29, 2014. Address correspondence to Dr Blumcke, Institute of Neuropathology, University Hospital Erlangen, Schwabachanlage 6, 91054 Erlangen, Germany. E-mail: [email protected] From the 1Department of Neuropathology, University Hospital Erlangen, Erlangen; 2Department of Virology, University Hospital Erlangen, Erlangen; 3 Bethel Epilepsy Center, Mara Hospital, Bielefeld; 4Department of Neurosurgery, Bielefeld Evangelical Hospital, Bielefeld; 5Department of Neurosurgery, University Hospital Erlangen, Erlangen; and 6Department of Pathology, University Hospital Erlangen, Erlangen, Germany
C 2014 American Neurological Association 312 V
Coras et al: No HPV Infection in FCDIIb
reports of HPV16 infection in formalin-fixed and paraffin-embedded (FFPE) brain tissue samples obtained from epilepsy patients with FCDIIb came as a surprise.12,13 Interestingly, the authors did not search for HPV infection by consensus polymerase chain reaction (PCR) assays for high-risk HPV types, but used assays designed to specifically detect HPV16 DNA or mRNA only. Using a PCR protocol designed to amplify the complete E6 coding region of HPV16 consisting of 477 base pairs (bp) from FFPE samples, Chen et al reported HPV16 DNA in 11 randomly selected FCDIIb specimens, and that 12 partially overlapping samples were also positive for HPV16 E6 mRNA.12 Immunohistochemistry showed labeling of the E6 epitope in cytoplasm of balloon cells, a pathognomic cell population in FCDIIb. A similar observation was reported briefly thereafter, detecting viral DNA by PCR amplification and viral protein by immunoreactivity for HPV16 as well as cytomegalovirus, herpes simplex virus 1, and human herpes virus 6B in 18 of 20 FFPE tissue specimens of FCDIIb.13 FCDs are frequent malformations of cortical development causing drug-resistant focal epilepsies.14–16 The International League against Epilepsy (ILAE) has proposed a first consensus classification system of FCDs based on the definition of distinct clinicopathological subtypes.17 According to this scheme, FCDIIb is neuropathologically characterized by enlarged dysmorphic neurons and balloon cells. Using magnetic resonance brain imaging, most patients with FCDIIb present with a transmantle sign,18 suggesting a maldevelopmental origin secondary to abnormal neuronal and glial proliferation or apoptosis.15 However, the specific molecular pathogenesis of FCDIIb has not been clarified yet. In our experience with the histopathological diagnosis of FCDIIb, multiple “isolated” FCDIIb lesions are rare, with only 1 case reported in the literature so far,19 which may be compatible with a viral etiopathogenesis. FCD-typic cellular abnormalities, that is, dysmorphic neurons and balloon cells, can be detected also in hemimegalencephaly (HME)16 or cortical tubers in patients suffering from tuberous sclerosis complex (TSC),20 and molecular analysis has provided evidence for a constitutive activation of the mTOR signaling pathway in FCDIIb, HME, and TSC.7–11,21–25 Activation of mTORC1 was detected in the same FCDIIb specimens reported to have HPV16 E6 infection.12 Nevertheless, we were not aware of cases with neoplastic transformation from FCDIIb into malignant brain tumors, despite the carcinogenic risk of E6 and E7 oncoproteins for high-risk HPV types. Appreciating the considerable interest in HPV biology as well as significance for FCD treatment and prevention,26 we February 2015
tried to confirm HPV16 detection in FCDIIb using HPV consensus PCR as well as the previously published PCR protocols for HPV16 in a series of 14 fresh-frozen surgical FCDIIb samples.
Patients and Methods Patients Included in the Study and Histopathological Examination of Surgical Brain Specimens We selected 14 consecutive patients submitted to epilepsy surgery at the Erlangen University Hospital and the BielefeldBethel Epilepsy Center (Table 1), in which the histopathological examination of surgical brain specimens confirmed the diagnosis of FCDIIb. Drug-resistant focal epilepsy was diagnosed by preoperative evaluation in all patients. The mean duration of epilepsy was 12 years, ranging from 1 to 30 years. The average age at surgery was 17.5 years, ranging from 4 to 43 years. All human tissues were obtained and processed in accordance with our approved institutional review board protocol. En bloc resected surgical specimens were carefully orientated and cut perpendicular to the gyral pattern into 5mmthick slices. According to our laboratory tissue procurement protocol, alternating samples were either snap frozen in liquid nitrogen and stored until further use (see below) or fixed overnight in 10% formalin and routinely processed into liquid paraffin. Paraffin-embedded sections were cut at 4mm with a microtome (Microm, Heidelberg, Germany), and mounted on positively charged slides (Superfrost; Menzel-Glaser, Braunschweig, Germany). Each specimen was microscopically reviewed by 2 neuropathologists (I.B. and R.C.) and classified by consensus definitions of the ILAE17 using hematoxylin and eosin as well as Cresyl violet (Nissl)–Luxol fast blue stainings. Histopathological examination included also the following panel of antibodies: MAP2 (microtubule-associated protein 2, 1:100, clone c, courtesy of Dr Riederer), NeuN27 (neuronal nuclei, 1:1,000, clone A-60; Millipore, Billerica, MA), nonphosphorylated neurofilament H (1:100, clone SMI-32; Covance, Emeryville, CA), GFAP (glial fibrillary acidic protein, 1:800, clone 6F2; Dako, Glostrup, Denmark), and vimentin (1:500, clone 3B4, Dako). Immunohistochemical stainings were performed with a semiautomated Ventana Benchmark Ultra XT machine following the manufacturer’s recommendations (Roche Diagnostics, Mannheim, Germany).
Use of Antibodies for the Immunohistochemical Detection of HPV Antigens To detect HPV antigens in FFPE human FCDIIb specimens, we used the following panel of antibodies: 1. Monoclonal mouse immunoglobulin G (IgG) antibody C1P5 (IL 61105; Thermo Scientific, Waltham, MA). The immunogen was gel-purified HPV-18 E6-beta galactosidase fusion. The product data sheet described an antibody reaction with HPV16 and 18 E6 in virus samples. Suggested application was specified for immunohistochemistry
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TABLE 1. Patients Included in This Study
ID
Gender
Lobe
Side
Age at Operation, yr
Age at Onset, yr
Duration, yr
Maximal Seizures/Month
1
M
Parietal
L
36
7
29
6
2
M
Temporoparieto-occipital
L
4
1
3
450
3
M
Temporoparietal
L
4
1
3
300
4
M
Temporo-occipital
R
5
1
4
1
5
F
Frontal
L
32
15
17
150
6
M
Frontal
R
24
1
23
200
7
F
Occipital
L
37
7
30
150
8
F
Frontal
R
11
4
7
300
9
F
Frontal
R
10
7
3
120
10
F
Occipital
L
16
3
13
16
11
F
Frontal
L
10
7
3
600
12
M
Frontal
R
6
5
1
900
13
M
Frontal
L
7
5
2
900
14
F
Frontal
R
43
13
30
150
F 5 female; L 5 left hemisphere; M 5 male; R 5 right hemisphere.
(paraffin; 1:10–1:2,000), immunoprecipitation (1:10–500), and Western blotting (1:100–2,000). We used this antibody for immunohistochemistry at a dilution of 1:500 and 1:1,000. This antibody was also used by Chen et al.12 2. Monoclonal mouse IgG antibody K1H8 supplied by Dako. The product data sheet described the antibody to react with a nonconformational internal linear epitope of a major capsid protein of HPV1, which is broadly expressed among different HPV subtypes including 6, 11, 16, 18, 31, 33, 52, 51, 52, 56, and 58 (confirmed by Southern blot hybridization). Suggested application was specified for immunohistochemistry (paraffin), with positive immunostaining confined to nuclei of infected cells. Furthermore, we tested antibodies directed against p16 as supplied by Santa Cruz Biotechnology (Santa Cruz, CA), which were raised against full-length recombinant p16 of human origin (clone JC8), and antibodies detecting endogenous levels of ribosomal protein S6 phosphorylated at serine 235 and serine 236 (pS6; Cell Signaling Technology, Danvers, MA). All antibody stainings described here were performed on 4mm thin paraffin sections using the histopathology protocol and a Ventana Benchmark semiautomated staining machine as described above.
PCR All 14 snap-frozen tissue specimens were cut at a cryostat (CM1950; Leica Biosystems, Wetzlar, Germany) and microscopically reviewed by experienced neuropathologists (R.C. and I.B.). Lesional FCDIIb tissue was confirmed in all cases. DNA
314
extraction from snap-frozen brain samples was performed with MagNaPure DNA Large Volume Kits (Roche Diagnostics). The efficiency of the extraction was controlled by a real-time PCR assay targeting the human albumin gene and by an endpoint PCR assay amplifying a 536bp fragment from the human ßglobin gene. For the detection of HPV DNA, 4 different endpoint PCR assays with detection by gel electrophoresis were used (for primer and probe sequences, see Table 2). Two assays were based on primers targeting sequences that are conserved among a broad range of HPV types, including HPV16. These primers amplify fragments of 461bp and 220bp, respectively, from the E1 gene.28 The 2 other assays were specific for HPV16 and amplify a 151bp fragment from the L1 gene and a 477bp fragment containing the complete E6 coding sequence of HPV16. The first 3 assays were established for routine detection and typing of HPV DNA in the Virology Department of the University Hospital Erlangen (founded by Dr Harald zur Hausen); the fourth assay is the one described by Chen et al.12
Results All 14 snap-frozen FCDIIb brain samples tested negative in the 4 PCR assays used here to detect HPV DNA, including the assay described by Chen et al in their paper reporting the detection of HPV16 DNA in FCDIIb.12 The protocols used by Liu et al were not specified.13 The presence of sufficient amounts of cellular DNA in samples was ascertained by 2 independent assays. A real-time PCR assay for the albumin gene detected between 2,100 and 23,000 Volume 77, No. 2
Coras et al: No HPV Infection in FCDIIb
TABLE 2. Sequences of Primers and Probes for HPV Detection and Controls
Primer Name CP4 CP5 PPF1 PPR2 16-1 16-2 16-E6F 16-E6R BG1 BG2 0
5 Alb 0
3 Alb Alb-Probe
Sequence
Target
50 -ATG GTA CAR TGG GCA TWT GA-30
HPV L1 (type-common)
0
5 -GAG GYT GCA ACC AAA AMT GRC-3
0
HPV L1 (type-common)
0
5 -AAC AAT GTG TAG ACA TTA TAA ACG AGC-3 0
5 -ATT AAA CTC ATT CCA AAA TAT GA-3 0
5 -TGC TAG TGC TTA TGC AGC AA-3 0
0
HPV-16L1 (type-specific)
5 -ATT TAC TGC AAC ATT GGT AC-3
HPV-16 L1 (type-specific)
0
5 -ATG CAC CCA AAG AGA ACT GCA-3
0
HPV-16E6 (type-specific)
0
0
5 -AAT ACA GCT GGG TTT CTC TAC G 23 0
5 -GGT TGG CCA ATC TAC TCC CAG G-3 5 -GCT CAC TCA GTG TGG CAA AG-3 0
0
0
HPV-16 E6 (type-specific) Human ß-globin gene Human ß-globin gene
0
5 -GTG AAC AGG CGA CCA TGC T-3 0
HPVL1 (type-common) HPVL1 (type-common)
0
0
0
0
Human albumin gene 0
5 -GCA TGG AAG GTG AAT GTT TCA G-3 0
5 -FAM-TCA GCT CTG GAA GTC GAT GAA ACA TAC GTT C-TAMRA
Human albumin gene Human albumin gene
HPV 5 human papillomavirus.
copies of cellular DNA per 10 ml of extracted DNA, and an endpoint PCR assay for the human ß-globin gene targeted a DNA fragment longer than all HPV PCR assays. Positive controls for HPV DNA detection included HPV16 plasmid DNA, HeLa cells containing HPV18 DNA, and clinical samples positive for HPV16 and various other HPV types. With the consensus PCR assays, all positive controls regardless of the HPV type showed positive results. Using HPV16-specific primer sets, only HPV16positive controls showed positive results, whereas controls positive for other HPV types were negative. Results of 1 representative PCR run are shown in Figure 1. Immunohistochemical staining with the antibody reported by Chen et al (C1P5) and directed against the
E6 epitope revealed weak labeling of cytoplasm from normal neurons and glial cells, as well as balloon cells and dysmorphic neurons in FCDIIb lesions (Fig 2). No specific labeling was observed with the antibody K1H8. We also detected immunolabeling of balloon cells with antibodies directed against the p16 and pS6 (ser235/ ser236) epitopes (Fig 3) using HPV-infected cervical tissue as positive control. Archival FFPE material from a TSC patient showed staining patterns similar to those described for FCDIIb (see Fig 3). The use of immunofluorescence staining protocols revealed bright autofluorescence of intracellular lipofuscin deposits at different excitation wavelengths in neuronal profiles (see Fig 2B, C), which rendered any interpretation of specific antibody labeling difficult. However, we could not identify specific labeling product in the nucleus of labeled cellular profiles. We also performed Western blotting to confirm the specificity of the C1P5 antibody using HPV16-infected cell cultures but failed to detect any reactivity (data not shown).
FIGURE 1: No evidence for viral infection with human papillomavirus 16 (HPV16) E6 DNA (477bp fragment) in surgical human brain specimens from patients with focal cortical dysplasia type IIb (FCDIIb). Lanes 1–10: FCDIIb brain specimens; lanes 11 and 16: no template controls; lanes 12 and 13: HPV16-positive cervical samples; lane 14: HPV11positive laryngeal papilloma sample; lane 15: HPV33positive cervical sample; lane M: size marker (100bp ladder).
Discussion
February 2015
None of our applied PCR protocols was able to demonstrate HPV16 DNA nor other HPV infections in surgical FCDIIb samples. Because we did not find any evidence for the presence of HPV16 DNA, we did not try to reproduce the HPV16 mRNA assay. In the absence of viral DNA, expression of mRNA from exactly the same 315
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FIGURE 2: Immunohistochemistry revealed unspecific labeling of the E6 epitope in human surgical brain specimens obtained from patients with focal cortical dysplasia type IIb (FCDIIb). (A) Immunohistochemistry using the C1P5 antibody supplied by Thermo Scientific and a semiautomated staining procedure with diaminobenzidine as brownish chromogen (Ventana Ultra) and hematoxylin as bluish counterstaining. Note cytoplasmic labeling of a dysmorphic neuron and glial cell in the FCDIIb specimen. (B, C) Autofluorescence is a common pitfall in human samples when using immunohistochemistry with fluorescent dyes. The same specimen shown in A revealed a neuronal profile with punctate lipofuscin granules around the nucleus, autofluorescent in B and C. (D) Bright light microscopic image at the same magnification and aperture as shown in B and C. (E) Balloon cells were also immunoreactive for the C1P5 antibody, as well as the glial cell profile depicted in A. Note that the nucleus is always spared from immunolabeling. (F) Clone K1H8 (Dako) does not show any specific labeling in a FCDIIb specimen (formalin-fixed and paraffin-embedded). Scale bar 5 20mm, applies to all parts.
region of the viral genome is impossible. An oncoproteinlike E6 staining was made visible by immunohistochemistry using FFPE human brain sections, but antibody labeling was not detectable in the cell nucleus (see Fig 2). Our data contradict 2 previous reports and require, therefore, careful consideration of differences in applied laboratory protocols and use of surgical human brain tissue. Chen and colleagues reported the detection of the HPV16 E6 protein, HPV16 DNA, and HPV16 mRNA in 50 of 50 surgical FCDIIb brain samples and proposed transplacental HPV16 infection as the underlying pathomechanism in FCDIIb.12 Although their data were corroborated with complementary methods, we could not
316
confirm the presence of HPV16 infection in our series of FCDIIb. Different methodological strategies were used in both studies, which may help to explain these contradictory results. Detection of HPV16 DNA was described in FFPE samples using a PCR assay targeting a large 477bp fragment covering the whole HPV16 E6 reading frame. In our clinicodiagnostic experience, however, amplification of viral DNA fragments >250bp are often unsuccessful in FFPE samples or result in unspecific template amplification. We chose snap-frozen, unfixed brain samples from histopathologically confirmed FCDIIb samples and applied complementary PCR assays with target sizes ranging from 151 to 477bp. With these protocols, we were not able to detect HPV16 DNA or DNA of other
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Coras et al: No HPV Infection in FCDIIb
FIGURE 3: Immunohistochemistry for p16, E6, and pS6 epitopes in human surgical brain specimens obtained from patients with focal cortical dysplasia type IIb (FCDIIb) and cortical tubers. (A–C) Immunohistochemistry in human papillomavirus–infected cervical intraepithelial neoplasia III (CIN III ) for p16 (positive, A), E6 (positive in cells of basal layer, arrow in B), and pS6 (weak staining in C). (D–F) FCDIIb. Balloon cells were consistently labeled with antibodies directed against p16 (D), E6 (E), and pS6 (F). (G–I) Cortical tuber in tuberous sclerosis complex (TSC). Balloon cells were consistently labeled with antibodies directed against p16 (G), E6 (H), and pS6 (I). Scale bar in C 5 100mm, applies also to A, B; scale bar in I 5 100mm, applies also to D–H.
HPV types in any of our 14 samples. Lack of HPV DNA detection in well-characterized FCDIIb brain samples with multiple assays covering a broad range of different HPV PCR assays also contradicts a report from China about the detection of HPV16 DNA in FCDIIb.13 However, the latter report was brief and specified neither applied PCR assays nor primer sequences and antibodies to allow any in-depth comparison with our presented protocols. The interpretation of unexpected immunohistochemical staining patterns and new findings need careful consideration when using FFPE brain specimens, as this methodology has its technical pitfalls.29 Reasons are multilayered and include fixation procedures, antibody clones, and their dilution.29 A plethora of various antiFebruary 2015
bodies can be purchased worldwide, and specification sheets claim positive staining results in human brain FFPE specimens. The antibody’s sensitivity is usually less well documented and needs corroboration for any specific fixation of brain tissue, embedding, and staining protocol in each laboratory. An antibody’s signal-to-noise staining ratio as well as the subcellular targets, whether membrane-bound, within cytoplasm, or within the nucleus, needs microscopic validation. As an example, we used the same antibody purchased by Chen et al and detected immunoreactivity in balloon cells, dysmorphic and hypoxic neurons, and a subpopulation of oligodendrocytes in FCDIIb and cortical tubers (see Figs 2 and 3). We concluded from our staining results that C1P5 antibody labeling was unspecific. Another possibility for 317
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unspecific cellular labeling signals is lipofuscin accumulation in adult human brain tissue when using fluorescent dyes for antibody detection (see Fig 2B, C). Liu and coworkers13 described cellular immunolabeling for HPV16, cytomegalovirus (CMV), human herpes virus 6B, and herpes simplex virus 1 (HSV1) with a low signal-to-noise ratio of their antibody staining allocated to the cytoplasm rather than showing intranuclear viral inclusions, such as Cowdry bodies in HSV or “owl eye” inclusions in CMV. Because many adult human neurons present with dotlike lipofuscin accumulation within their cytoplasm,30 nonspecific autofluorescent signals can be detected at different excitation and emission wavelengths. The mode of HPV transmission remains an open question in the proposed HPV16 etiology of FCDIIb. Chen et al argued that “transplacental spread of HPV16 is a plausible mechanism for entry into the brain during early cortical development” based on 1 study reporting a transplacental HPV transmission rate of 12.2% in women with HPV-associated genital lesions20 and another study reporting an estimated HPV prevalence of 26.8% in women between 14 and 59 years old in the USA.31 However, in the first study, from Brazil, none of the 11 HPV-infected newborns was positive for HPV16, and in the second study HPV16 was detected in only 1.5% of the samples, thus representing
No Evidence for Human Papillomavirus Infection in Focal Cortical Dysplasia IIb Roland Coras, MD,1 Klaus Korn, MD,2 Christian G. Bien, MD,3 Thilo Kalbhenn, MD,4 Karl R€ ossler, MD,5 Katja Kobow, PhD,1 Johannes Giedl, MD,6 Bernhard Fleckenstein, MD, PhD,2 and Ingmar Blumcke, MD1 Objective: The etiology of focal cortical dysplasia type IIb (FCDIIb) remains enigmatic in patients suffering from drug-resistant epilepsy, and an aberrant activation of the mammalian target of rapamycin complex 1 signaling pathway (mTORC1) was detected in this developmental brain malformation. Recently, the human papillomavirus (HPV) oncoprotein E6 has been identified as a potent activator of mTORC1, and HPV16 E6 has been described to persist in balloon cells obtained from surgical FCDIIb specimens. Although this observation was replicated by an independent second report, it contradicts current knowledge of HPV biology. HPV infects the squamous or mucocutaneous epithelium; hematogenic spread into other tissues has not been observed. In addition, brain carcinogenesis has never been reported in FCDIIb patients. Herein, we have tried to confirm 2 previous reports of HPV16 E6 infection using an independent series of 14 surgical specimens with histopathologically confirmed FCDIIb. Methods: Snap-frozen FCDIIb specimens were tested for HPV DNA using the primer set for amplification of the complete E6 reading frame of HPV16 and 3 other sets of primers (2 consensus primer sets detecting multiple HPV genotypes, and another primer set specifically used for HPV16). Furthermore, formalin-fixed and paraffin-embedded histopathological preparations were immunohistochemically analyzed using previously described antibodies directed against the HPV E6 oncoprotein. Results: All 14 FCDIIb specimens were negative for HPV DNA with all 4 primer sets. Antibodies directed against the HPV E6 epitope showed weak labeling of cytoplasm in balloon cells, as previously described in FCDIIb, but also in other cell populations. Interpretation: Our data did not confirm previously reported evidence for HPV16 detection in FCDIIb. ANN NEUROL 2015;77:312–319
H
uman papillomaviruses (HPVs) belong to a DNA virus family capable of infecting keratinocytes of the skin or mucous membranes. Their causative role in cervical carcinomas was first postulated by Harald zur Hausen in 1976, and since then has been studied and confirmed in many human cancers, including squamous cell carcinomas of the head and neck.1,2 More than 150 different HPV types have been reported, and at least 15 types are considered as carcinogenic.3 Carcinogenesis is conveyed by E6 and E7 oncoproteins of high-risk HPV types, especially HPV16 and HPV18. E6 binds to p53,4 and E7 to the retinoblastoma tumor suppressor gene product
pRB,5 thereby effecting similar tumor transformation pathways. More recently, HPV16 E6 expression was shown to also cause an increase in mammalian target of rapamycin complex 1 signaling pathway (mTORC1) activity through an enhanced phosphorylation of mTOR and activation of downstream signaling pathways (ie, Akt and S6K).6 mTOR activation is considered an important molecular hallmark also in focal cortical dysplasia type IIb (FCDIIb), as shown either by genetic TSC1 analysis or immunohistochemistry for activated downstream targets.7–11 Considering the well-characterized biology and epidemiology of high-risk HPV genotypes, 2 recent
View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24328 Received Jul 9, 2014, and in revised form Nov 24, 2014. Accepted for publication Nov 29, 2014. Address correspondence to Dr Blumcke, Institute of Neuropathology, University Hospital Erlangen, Schwabachanlage 6, 91054 Erlangen, Germany. E-mail: [email protected] From the 1Department of Neuropathology, University Hospital Erlangen, Erlangen; 2Department of Virology, University Hospital Erlangen, Erlangen; 3 Bethel Epilepsy Center, Mara Hospital, Bielefeld; 4Department of Neurosurgery, Bielefeld Evangelical Hospital, Bielefeld; 5Department of Neurosurgery, University Hospital Erlangen, Erlangen; and 6Department of Pathology, University Hospital Erlangen, Erlangen, Germany
C 2014 American Neurological Association 312 V
Coras et al: No HPV Infection in FCDIIb
reports of HPV16 infection in formalin-fixed and paraffin-embedded (FFPE) brain tissue samples obtained from epilepsy patients with FCDIIb came as a surprise.12,13 Interestingly, the authors did not search for HPV infection by consensus polymerase chain reaction (PCR) assays for high-risk HPV types, but used assays designed to specifically detect HPV16 DNA or mRNA only. Using a PCR protocol designed to amplify the complete E6 coding region of HPV16 consisting of 477 base pairs (bp) from FFPE samples, Chen et al reported HPV16 DNA in 11 randomly selected FCDIIb specimens, and that 12 partially overlapping samples were also positive for HPV16 E6 mRNA.12 Immunohistochemistry showed labeling of the E6 epitope in cytoplasm of balloon cells, a pathognomic cell population in FCDIIb. A similar observation was reported briefly thereafter, detecting viral DNA by PCR amplification and viral protein by immunoreactivity for HPV16 as well as cytomegalovirus, herpes simplex virus 1, and human herpes virus 6B in 18 of 20 FFPE tissue specimens of FCDIIb.13 FCDs are frequent malformations of cortical development causing drug-resistant focal epilepsies.14–16 The International League against Epilepsy (ILAE) has proposed a first consensus classification system of FCDs based on the definition of distinct clinicopathological subtypes.17 According to this scheme, FCDIIb is neuropathologically characterized by enlarged dysmorphic neurons and balloon cells. Using magnetic resonance brain imaging, most patients with FCDIIb present with a transmantle sign,18 suggesting a maldevelopmental origin secondary to abnormal neuronal and glial proliferation or apoptosis.15 However, the specific molecular pathogenesis of FCDIIb has not been clarified yet. In our experience with the histopathological diagnosis of FCDIIb, multiple “isolated” FCDIIb lesions are rare, with only 1 case reported in the literature so far,19 which may be compatible with a viral etiopathogenesis. FCD-typic cellular abnormalities, that is, dysmorphic neurons and balloon cells, can be detected also in hemimegalencephaly (HME)16 or cortical tubers in patients suffering from tuberous sclerosis complex (TSC),20 and molecular analysis has provided evidence for a constitutive activation of the mTOR signaling pathway in FCDIIb, HME, and TSC.7–11,21–25 Activation of mTORC1 was detected in the same FCDIIb specimens reported to have HPV16 E6 infection.12 Nevertheless, we were not aware of cases with neoplastic transformation from FCDIIb into malignant brain tumors, despite the carcinogenic risk of E6 and E7 oncoproteins for high-risk HPV types. Appreciating the considerable interest in HPV biology as well as significance for FCD treatment and prevention,26 we February 2015
tried to confirm HPV16 detection in FCDIIb using HPV consensus PCR as well as the previously published PCR protocols for HPV16 in a series of 14 fresh-frozen surgical FCDIIb samples.
Patients and Methods Patients Included in the Study and Histopathological Examination of Surgical Brain Specimens We selected 14 consecutive patients submitted to epilepsy surgery at the Erlangen University Hospital and the BielefeldBethel Epilepsy Center (Table 1), in which the histopathological examination of surgical brain specimens confirmed the diagnosis of FCDIIb. Drug-resistant focal epilepsy was diagnosed by preoperative evaluation in all patients. The mean duration of epilepsy was 12 years, ranging from 1 to 30 years. The average age at surgery was 17.5 years, ranging from 4 to 43 years. All human tissues were obtained and processed in accordance with our approved institutional review board protocol. En bloc resected surgical specimens were carefully orientated and cut perpendicular to the gyral pattern into 5mmthick slices. According to our laboratory tissue procurement protocol, alternating samples were either snap frozen in liquid nitrogen and stored until further use (see below) or fixed overnight in 10% formalin and routinely processed into liquid paraffin. Paraffin-embedded sections were cut at 4mm with a microtome (Microm, Heidelberg, Germany), and mounted on positively charged slides (Superfrost; Menzel-Glaser, Braunschweig, Germany). Each specimen was microscopically reviewed by 2 neuropathologists (I.B. and R.C.) and classified by consensus definitions of the ILAE17 using hematoxylin and eosin as well as Cresyl violet (Nissl)–Luxol fast blue stainings. Histopathological examination included also the following panel of antibodies: MAP2 (microtubule-associated protein 2, 1:100, clone c, courtesy of Dr Riederer), NeuN27 (neuronal nuclei, 1:1,000, clone A-60; Millipore, Billerica, MA), nonphosphorylated neurofilament H (1:100, clone SMI-32; Covance, Emeryville, CA), GFAP (glial fibrillary acidic protein, 1:800, clone 6F2; Dako, Glostrup, Denmark), and vimentin (1:500, clone 3B4, Dako). Immunohistochemical stainings were performed with a semiautomated Ventana Benchmark Ultra XT machine following the manufacturer’s recommendations (Roche Diagnostics, Mannheim, Germany).
Use of Antibodies for the Immunohistochemical Detection of HPV Antigens To detect HPV antigens in FFPE human FCDIIb specimens, we used the following panel of antibodies: 1. Monoclonal mouse immunoglobulin G (IgG) antibody C1P5 (IL 61105; Thermo Scientific, Waltham, MA). The immunogen was gel-purified HPV-18 E6-beta galactosidase fusion. The product data sheet described an antibody reaction with HPV16 and 18 E6 in virus samples. Suggested application was specified for immunohistochemistry
313
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of Neurology
TABLE 1. Patients Included in This Study
ID
Gender
Lobe
Side
Age at Operation, yr
Age at Onset, yr
Duration, yr
Maximal Seizures/Month
1
M
Parietal
L
36
7
29
6
2
M
Temporoparieto-occipital
L
4
1
3
450
3
M
Temporoparietal
L
4
1
3
300
4
M
Temporo-occipital
R
5
1
4
1
5
F
Frontal
L
32
15
17
150
6
M
Frontal
R
24
1
23
200
7
F
Occipital
L
37
7
30
150
8
F
Frontal
R
11
4
7
300
9
F
Frontal
R
10
7
3
120
10
F
Occipital
L
16
3
13
16
11
F
Frontal
L
10
7
3
600
12
M
Frontal
R
6
5
1
900
13
M
Frontal
L
7
5
2
900
14
F
Frontal
R
43
13
30
150
F 5 female; L 5 left hemisphere; M 5 male; R 5 right hemisphere.
(paraffin; 1:10–1:2,000), immunoprecipitation (1:10–500), and Western blotting (1:100–2,000). We used this antibody for immunohistochemistry at a dilution of 1:500 and 1:1,000. This antibody was also used by Chen et al.12 2. Monoclonal mouse IgG antibody K1H8 supplied by Dako. The product data sheet described the antibody to react with a nonconformational internal linear epitope of a major capsid protein of HPV1, which is broadly expressed among different HPV subtypes including 6, 11, 16, 18, 31, 33, 52, 51, 52, 56, and 58 (confirmed by Southern blot hybridization). Suggested application was specified for immunohistochemistry (paraffin), with positive immunostaining confined to nuclei of infected cells. Furthermore, we tested antibodies directed against p16 as supplied by Santa Cruz Biotechnology (Santa Cruz, CA), which were raised against full-length recombinant p16 of human origin (clone JC8), and antibodies detecting endogenous levels of ribosomal protein S6 phosphorylated at serine 235 and serine 236 (pS6; Cell Signaling Technology, Danvers, MA). All antibody stainings described here were performed on 4mm thin paraffin sections using the histopathology protocol and a Ventana Benchmark semiautomated staining machine as described above.
PCR All 14 snap-frozen tissue specimens were cut at a cryostat (CM1950; Leica Biosystems, Wetzlar, Germany) and microscopically reviewed by experienced neuropathologists (R.C. and I.B.). Lesional FCDIIb tissue was confirmed in all cases. DNA
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extraction from snap-frozen brain samples was performed with MagNaPure DNA Large Volume Kits (Roche Diagnostics). The efficiency of the extraction was controlled by a real-time PCR assay targeting the human albumin gene and by an endpoint PCR assay amplifying a 536bp fragment from the human ßglobin gene. For the detection of HPV DNA, 4 different endpoint PCR assays with detection by gel electrophoresis were used (for primer and probe sequences, see Table 2). Two assays were based on primers targeting sequences that are conserved among a broad range of HPV types, including HPV16. These primers amplify fragments of 461bp and 220bp, respectively, from the E1 gene.28 The 2 other assays were specific for HPV16 and amplify a 151bp fragment from the L1 gene and a 477bp fragment containing the complete E6 coding sequence of HPV16. The first 3 assays were established for routine detection and typing of HPV DNA in the Virology Department of the University Hospital Erlangen (founded by Dr Harald zur Hausen); the fourth assay is the one described by Chen et al.12
Results All 14 snap-frozen FCDIIb brain samples tested negative in the 4 PCR assays used here to detect HPV DNA, including the assay described by Chen et al in their paper reporting the detection of HPV16 DNA in FCDIIb.12 The protocols used by Liu et al were not specified.13 The presence of sufficient amounts of cellular DNA in samples was ascertained by 2 independent assays. A real-time PCR assay for the albumin gene detected between 2,100 and 23,000 Volume 77, No. 2
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TABLE 2. Sequences of Primers and Probes for HPV Detection and Controls
Primer Name CP4 CP5 PPF1 PPR2 16-1 16-2 16-E6F 16-E6R BG1 BG2 0
5 Alb 0
3 Alb Alb-Probe
Sequence
Target
50 -ATG GTA CAR TGG GCA TWT GA-30
HPV L1 (type-common)
0
5 -GAG GYT GCA ACC AAA AMT GRC-3
0
HPV L1 (type-common)
0
5 -AAC AAT GTG TAG ACA TTA TAA ACG AGC-3 0
5 -ATT AAA CTC ATT CCA AAA TAT GA-3 0
5 -TGC TAG TGC TTA TGC AGC AA-3 0
0
HPV-16L1 (type-specific)
5 -ATT TAC TGC AAC ATT GGT AC-3
HPV-16 L1 (type-specific)
0
5 -ATG CAC CCA AAG AGA ACT GCA-3
0
HPV-16E6 (type-specific)
0
0
5 -AAT ACA GCT GGG TTT CTC TAC G 23 0
5 -GGT TGG CCA ATC TAC TCC CAG G-3 5 -GCT CAC TCA GTG TGG CAA AG-3 0
0
0
HPV-16 E6 (type-specific) Human ß-globin gene Human ß-globin gene
0
5 -GTG AAC AGG CGA CCA TGC T-3 0
HPVL1 (type-common) HPVL1 (type-common)
0
0
0
0
Human albumin gene 0
5 -GCA TGG AAG GTG AAT GTT TCA G-3 0
5 -FAM-TCA GCT CTG GAA GTC GAT GAA ACA TAC GTT C-TAMRA
Human albumin gene Human albumin gene
HPV 5 human papillomavirus.
copies of cellular DNA per 10 ml of extracted DNA, and an endpoint PCR assay for the human ß-globin gene targeted a DNA fragment longer than all HPV PCR assays. Positive controls for HPV DNA detection included HPV16 plasmid DNA, HeLa cells containing HPV18 DNA, and clinical samples positive for HPV16 and various other HPV types. With the consensus PCR assays, all positive controls regardless of the HPV type showed positive results. Using HPV16-specific primer sets, only HPV16positive controls showed positive results, whereas controls positive for other HPV types were negative. Results of 1 representative PCR run are shown in Figure 1. Immunohistochemical staining with the antibody reported by Chen et al (C1P5) and directed against the
E6 epitope revealed weak labeling of cytoplasm from normal neurons and glial cells, as well as balloon cells and dysmorphic neurons in FCDIIb lesions (Fig 2). No specific labeling was observed with the antibody K1H8. We also detected immunolabeling of balloon cells with antibodies directed against the p16 and pS6 (ser235/ ser236) epitopes (Fig 3) using HPV-infected cervical tissue as positive control. Archival FFPE material from a TSC patient showed staining patterns similar to those described for FCDIIb (see Fig 3). The use of immunofluorescence staining protocols revealed bright autofluorescence of intracellular lipofuscin deposits at different excitation wavelengths in neuronal profiles (see Fig 2B, C), which rendered any interpretation of specific antibody labeling difficult. However, we could not identify specific labeling product in the nucleus of labeled cellular profiles. We also performed Western blotting to confirm the specificity of the C1P5 antibody using HPV16-infected cell cultures but failed to detect any reactivity (data not shown).
FIGURE 1: No evidence for viral infection with human papillomavirus 16 (HPV16) E6 DNA (477bp fragment) in surgical human brain specimens from patients with focal cortical dysplasia type IIb (FCDIIb). Lanes 1–10: FCDIIb brain specimens; lanes 11 and 16: no template controls; lanes 12 and 13: HPV16-positive cervical samples; lane 14: HPV11positive laryngeal papilloma sample; lane 15: HPV33positive cervical sample; lane M: size marker (100bp ladder).
Discussion
February 2015
None of our applied PCR protocols was able to demonstrate HPV16 DNA nor other HPV infections in surgical FCDIIb samples. Because we did not find any evidence for the presence of HPV16 DNA, we did not try to reproduce the HPV16 mRNA assay. In the absence of viral DNA, expression of mRNA from exactly the same 315
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FIGURE 2: Immunohistochemistry revealed unspecific labeling of the E6 epitope in human surgical brain specimens obtained from patients with focal cortical dysplasia type IIb (FCDIIb). (A) Immunohistochemistry using the C1P5 antibody supplied by Thermo Scientific and a semiautomated staining procedure with diaminobenzidine as brownish chromogen (Ventana Ultra) and hematoxylin as bluish counterstaining. Note cytoplasmic labeling of a dysmorphic neuron and glial cell in the FCDIIb specimen. (B, C) Autofluorescence is a common pitfall in human samples when using immunohistochemistry with fluorescent dyes. The same specimen shown in A revealed a neuronal profile with punctate lipofuscin granules around the nucleus, autofluorescent in B and C. (D) Bright light microscopic image at the same magnification and aperture as shown in B and C. (E) Balloon cells were also immunoreactive for the C1P5 antibody, as well as the glial cell profile depicted in A. Note that the nucleus is always spared from immunolabeling. (F) Clone K1H8 (Dako) does not show any specific labeling in a FCDIIb specimen (formalin-fixed and paraffin-embedded). Scale bar 5 20mm, applies to all parts.
region of the viral genome is impossible. An oncoproteinlike E6 staining was made visible by immunohistochemistry using FFPE human brain sections, but antibody labeling was not detectable in the cell nucleus (see Fig 2). Our data contradict 2 previous reports and require, therefore, careful consideration of differences in applied laboratory protocols and use of surgical human brain tissue. Chen and colleagues reported the detection of the HPV16 E6 protein, HPV16 DNA, and HPV16 mRNA in 50 of 50 surgical FCDIIb brain samples and proposed transplacental HPV16 infection as the underlying pathomechanism in FCDIIb.12 Although their data were corroborated with complementary methods, we could not
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confirm the presence of HPV16 infection in our series of FCDIIb. Different methodological strategies were used in both studies, which may help to explain these contradictory results. Detection of HPV16 DNA was described in FFPE samples using a PCR assay targeting a large 477bp fragment covering the whole HPV16 E6 reading frame. In our clinicodiagnostic experience, however, amplification of viral DNA fragments >250bp are often unsuccessful in FFPE samples or result in unspecific template amplification. We chose snap-frozen, unfixed brain samples from histopathologically confirmed FCDIIb samples and applied complementary PCR assays with target sizes ranging from 151 to 477bp. With these protocols, we were not able to detect HPV16 DNA or DNA of other
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FIGURE 3: Immunohistochemistry for p16, E6, and pS6 epitopes in human surgical brain specimens obtained from patients with focal cortical dysplasia type IIb (FCDIIb) and cortical tubers. (A–C) Immunohistochemistry in human papillomavirus–infected cervical intraepithelial neoplasia III (CIN III ) for p16 (positive, A), E6 (positive in cells of basal layer, arrow in B), and pS6 (weak staining in C). (D–F) FCDIIb. Balloon cells were consistently labeled with antibodies directed against p16 (D), E6 (E), and pS6 (F). (G–I) Cortical tuber in tuberous sclerosis complex (TSC). Balloon cells were consistently labeled with antibodies directed against p16 (G), E6 (H), and pS6 (I). Scale bar in C 5 100mm, applies also to A, B; scale bar in I 5 100mm, applies also to D–H.
HPV types in any of our 14 samples. Lack of HPV DNA detection in well-characterized FCDIIb brain samples with multiple assays covering a broad range of different HPV PCR assays also contradicts a report from China about the detection of HPV16 DNA in FCDIIb.13 However, the latter report was brief and specified neither applied PCR assays nor primer sequences and antibodies to allow any in-depth comparison with our presented protocols. The interpretation of unexpected immunohistochemical staining patterns and new findings need careful consideration when using FFPE brain specimens, as this methodology has its technical pitfalls.29 Reasons are multilayered and include fixation procedures, antibody clones, and their dilution.29 A plethora of various antiFebruary 2015
bodies can be purchased worldwide, and specification sheets claim positive staining results in human brain FFPE specimens. The antibody’s sensitivity is usually less well documented and needs corroboration for any specific fixation of brain tissue, embedding, and staining protocol in each laboratory. An antibody’s signal-to-noise staining ratio as well as the subcellular targets, whether membrane-bound, within cytoplasm, or within the nucleus, needs microscopic validation. As an example, we used the same antibody purchased by Chen et al and detected immunoreactivity in balloon cells, dysmorphic and hypoxic neurons, and a subpopulation of oligodendrocytes in FCDIIb and cortical tubers (see Figs 2 and 3). We concluded from our staining results that C1P5 antibody labeling was unspecific. Another possibility for 317
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unspecific cellular labeling signals is lipofuscin accumulation in adult human brain tissue when using fluorescent dyes for antibody detection (see Fig 2B, C). Liu and coworkers13 described cellular immunolabeling for HPV16, cytomegalovirus (CMV), human herpes virus 6B, and herpes simplex virus 1 (HSV1) with a low signal-to-noise ratio of their antibody staining allocated to the cytoplasm rather than showing intranuclear viral inclusions, such as Cowdry bodies in HSV or “owl eye” inclusions in CMV. Because many adult human neurons present with dotlike lipofuscin accumulation within their cytoplasm,30 nonspecific autofluorescent signals can be detected at different excitation and emission wavelengths. The mode of HPV transmission remains an open question in the proposed HPV16 etiology of FCDIIb. Chen et al argued that “transplacental spread of HPV16 is a plausible mechanism for entry into the brain during early cortical development” based on 1 study reporting a transplacental HPV transmission rate of 12.2% in women with HPV-associated genital lesions20 and another study reporting an estimated HPV prevalence of 26.8% in women between 14 and 59 years old in the USA.31 However, in the first study, from Brazil, none of the 11 HPV-infected newborns was positive for HPV16, and in the second study HPV16 was detected in only 1.5% of the samples, thus representing
