Giant cell arteritis is one of the most common causes of vasculitis involving the temporal artery. Other potential causes are Wegener granulomatosis, polyarteritis nodosa (PAN), and Buerger disease[1]. GCA generally affects individuals over 55 years of age (with a mean age at diagnosis of approximately 72 years)[2] with an annual incidence of approximately 18 per 100,000 in persons aged 50 years or older.

Histologically, GCA shows transmural inflammation with mixed inflammatory cell infiltrate mostly consisting of lymphocytes, histiocytes, plasma cells, occasional neutrophils and rarely eosinophils (Figure 1). This causes destruction of the vessel's internal elastic lamina which is best demonstrated with elastic stains such as Verhoeff-Van Gieson (Figure 2) or Movat pentachrome. The presence of giant cells, next to the elastic lamina in particular, is the classic and pathognomonic feature of GCA. However, this is present in only about 50% of biopsy-proven cases.

Infectious agents have long been considered as a possible etiology of GCA. The concept is that GCA represents a chronic inflammatory response, triggered by an infectious agent, with subsequent inappropriate tissue response to injury. However, studies looking for organisms have had conflicting results. Some have demonstrated organism DNA, such as Herpes Simplex Virus, while the vast majority have failed to demonstrate an association. It is speculated that the inflammatory response may be triggered by an infectious agent. If a connection between GCA and HPV infection were to be established, the potential clinical implications are great, as GCA, with its possible vision loss[3–5], polymyalgia rheumatica and even eventually ischemic stroke[6–9], might potentially be prevented by vaccination or other strategies.

Human papillomavirus (HPV) is an increasingly common human pathogen in recent decades. It is a mucosotropic virus which is not thought of as spreading systemically. However, HPV genotype 16 has been found integrated into the genome of bacteria isolated from cervical cancer biopsies, and there is also published data showing HPV viral particles within peripheral nerves and small vascular endothelial cells adjacent to oral and cervical cancers as demonstrated by transmission electron microscopy[7, 10]. In this study, we sought to determine if there is an association between GCA and HPV.

With approval by the Washington University Human Research Protection Office (HRPO), we searched the Copath database of Barnes Jewish Hospital for temporal artery biopsy specimens from 1995 to 2008 and retrieved all specimens for which material was available. We identified approximately 60 cases, of which 43, (22 histologically positive and 21 histologically negative) had material available for review. There were five males and 17 females in the histologically positive GCA group with a mean age of 78.9 years. In the histologically negative group, there were four males and 17 females with a mean age of 67 years, and the control kidney vascular margin group had six males and nine females with a mean age of 38.2 years. There were no statistically significant difference in gender among the groups (p = 0.176). The clinical diagnosis of GCA was based on the criteria of the American College of Rheumatology[11]. However, for our study, we considered the gold standard to be histologic evidence of temporal arteritis. We also randomly selected 15 renal artery vascular resection margins from nephrectomy specimens in patients without any history of vasculitis for use as the negative controls.

Two methods were utilized to evaluate for HPV DNA. The first, INNO-LiPA HPV Genotyping Extra, was used to perform the testing in our research laboratory of the division of anatomic and molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis. All blocks were then submitted to an outside laboratory (CPA Laboratory, Louisville, KY) for independent preparation of genomic DNA and for testing employing the Cervista^TM HPV HR (Hologic, Madison, WI, USA) assay.

In both laboratories to avoid potential contamination, a maximum physical separation between the pre- and post-amplification steps was used. Separate pipettes and other lab materials were used as a part of good laboratory practice. The FFPE blocks were cut and processed under strict conditions to prevent DNA from being carried over from one case to the next during microtomy. Also a new blade was used for each case and the area was cleaned. Ice cubes used to cool blocks were discarded between cases.

With regards to the technical limitations of this study, it is well known that formalin fixation randomly fragments DNA in a duration-dependent manner, resulting in a partial degradation. The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation. Due to this degradation, FFPE tissue is not suitable for amplification of large DNA segments. Nevertheless, PCR amplification of segments ranging up to 1300 base pair has been reported. On the other hand, incubation at an elevated temperature after proteinase K digestion partially removes formalin cross-linking of the DNA, improving yield as well as DNA performance in assays. Furthermore, in one of the techniques used in this study (INNO-LiPA HPV Genotyping), short-PCR-fragment (SPF 10) primers are employed, which amplify a 65 base pair segment of target DNA and this testing is considered to be one of the most sensitive PCR assays for the detection of HPV DNA.

INNO-LiPA HPV Genotyping testing

The INNO-LiPA HPV Genotyping Extra is based on the principle of reverse hybridization. Part of the L1 region of the human papillomavirus (HPV) genome is amplified using short-PCR-fragment assay (SPF10 primers), and the resulting biotinylated amplicons are then denatured and hybridized with specific oligonucleotide probes.

An additional primer pair for the amplification of the human HLA-DPB1 gene is added to monitor sample quality and extraction. The length of the HLA-DPB1 fragment is 280 base pairs. All probes are immobilized as parallel lines on membrane strips. After hybridization and stringent washing, streptavidin-conjugated alkaline phosphatase is added, which binds to any biotinylated hybrid previously formed.

Incubation with BCIP (5-Bromo-4-Chloro-3’-Indolyphosphate p-Toluidine Salt)/NBT (Nitro-Blue Tetrazolium Chloride) chromogen yields a purple precipitate, and the results are visually interpreted using the reference guide provided. An amplification kit (INNO-LiPA HPV Genotyping Extra Amp) is used for standardized preparation of biotinylated amplified material. This amplification kit is based on the polymerase chain reaction (PCR) using SPF10 primers.

Amplification products are subsequently hybridized using a single typing strip on which 28 sequence-specific DNA probe lines and 4 control lines are fixed, which permits specific detection of 28 HPV genotypes, including all 18 high-risk genotypes, and 10 low-risk genotypes (HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74, and 82) as described by the manufacturer (Figure 3).

Among the 21 cases of GCA histologically negative biopsies, 5 were positive for HPV by INNO-LiPA HPV Genotyping Extra, of which 3 (60%) were genotype 16, one (20%) was genotype 6 and one (20%) was genotype 52. Cervista^TM HPV HR testing revealed positivity of Master Mix 3 for all 4 cases which were positive by INNO-LiPA HPV Genotyping Extra for high risk HPV. Case number 2 which was negative with INNO-LiPA was of low genomic DNA with Cervista method. As such, there was 100% agreement between the two testing methods on the histologically negative temporal artery biopsy cases.

Among the 15 nephrectomy vascular resection margins (controls), 3 were positive for HPV by INNO-LiPA HPV Genotyping Extra. Two cases showed genotype 16 (1 case of Wilms' tumor and 1 of acute and chronic pyelonephritis), and one genotype 6 (a case of papillary urothelial carcinoma). Cervista^TM HPV HR testing revealed positivity of Master Mix 3 for both cases which were positive for high risk HPV by INNO-LiPA HPV Genotyping Extra. As such, there was 100% agreement between the two testing methods on the 15 vascular margin control cases.

The association between HPV positivity and histologically confirmed GCA was statistically significant (p = 0.0013). There was also a statistically significant association between HPV positivity and Caucasian race (p = 0.0339). No other associations were statistically significant.

Giant cell (temporal) arteritis is a chronic vasculitis involving medium and large size arteries that typically affects individuals older than 50 years of age.[1, 3, 4] Although it usually affects the superficial temporal arteries, it can also affect the aorta, carotid, subclavian, vertebral, and iliac arteries. The classical picture of granulomatous inflammation with multinucleated giant cells is observed in approximately 50% of the patients.

Histologically, the disease progresses from minimal involvement of vessels, with only collections of lymphocytes confined to the internal or external elastic lamina or adventitia to a panarteritis with segmental areas of necrosis of the arterial wall and extensive destruction of the elastic laminae, a feature that can be clearly demonstrated with special stains for elastic fibers (Figures 1 and2).

The etiology of the inflammatory reaction in the GCA has not been identified. However, it has been demonstrated that there is a restricted clonal expansion of tissue-infiltrating T cells in these lesions, which suggests that they are reacting to a specific antigen located within the affected arterial wall which is thus eliciting the disease[13]. The most recent hypothesis regarding the etiology of GCA contends that a response to endothelial injury (maybe due to infection) leads to an inappropriate activation of T-cell-mediated immunity[14, 15]. The subsequent release of inflammatory mediators within the arterial vessel wall can attract macrophages which then become multinucleated giant cells, creating the characteristic histology of this disease and also leading to an oligoclonal expansion of T-cells directed against antigens within or near the elastic lamina. This cascade of events in due course results in vessel wall damage, intimal hyperplasia, and eventual stenotic occlusion.

Infectious agents have been considered in the past as the etiology of GCA. Elling et al., by indirect serological evidence, found high incidence of GCA within a population associated with Chlamydia pneumoniae, Mycoplasma pneumoniae , and Parvovirus B19[16]. Published data from Wagner et al.[15] and Powers et al.[17] reported a relationship between GCA, C. pneumonia and herpes simplex virus (HSV). However, further studies were unable to detect the presence of these infectious agents. Cooper et al.[14] and Cankovic and Zarbo[18], could not confirm the association between GCA and C. pneumonia, HSV, varicella zoster virus (VZV), Epstein–Barr virus (EBV), or human herpesvirus 7 (HHV7). In addition, Rodriguez-Pla et al.[19], Helweg-Larsen[20] and other authors[21–23] could not confirm the presence of herpes viruses, varicella zoster virus, parvovirus, or C. pneumoniae in temporal artery GCA. Due to this conflicting and contradictory data, no conclusive link has, to date, been demonstrated between a GCA and a specific infectious agent.

The role of human papillomavirus (HPV) in the development of cervical, head and neck, anal and skin cancers is well known. Molecular and epidemiological studies have shown that persistent HPV infection is the most important risk factor for cervical cancer[24, 25]. High risk HPV also has an important role in anogenital and oropharyngeal squamous cell carcinoma[26]. They are also implicated with skin cancer in individuals with epidermodysplasia verruciformis[27] and can induce a variety of proliferative lesions, such as warts and laryngeal papillomas. There are very few associations between HPV and non-neoplastic diseases.

Interestingly, Ma et al.[10] reported the presence of HPV type 16 in the genome of bacterial strains (Enterococcus , Staphylococcus , Bacillus and Corynebacterium) and demonstrated the HPV viral particles by transmission electron microscopy in those bacteria. In addition, HPV type 16 has been detected in peripheral nerves, and vascular endothelial cells[7]. Another possible pathway is for HPV to disseminate systemically. This has long been debated[28]. The role of viremia in the pathogenesis of HPV-related diseases is still unclear, although HPV DNA has been detected in peripheral blood in some studies, though in varying amounts[29–32].

In this study, we found that HPV DNA, of both high and low risk types, is present in formalin-fixed, paraffin-embedded temporal artery biopsy specimens, and in statistically significantly higher numbers in the arteritis specimens compared to histologically-negative temporal artery biopsies and nephrectomy vascular margin controls. The validity of these results was confirmed with exactly matching results by an outside laboratory that independently prepped DNA from the paraffin blocks and utilized a completely different detection method. Our results are admittedly surprising and bring many questions about what HPV's role, if any, would be in temporal arteritis. HPV has been associated with tumorigenesis, and many studies have investigated HPV-related modification of the immune system to establish infection. However, we are not aware of any literature citing HPV as an inciting agent for inflammatory diseases. Our findings do support the notion that HPV can disseminate systemically, but do not, by themselves, tell us anything specific about the pathophysiological relationship between the virus and temporal arteritis, if there even is one. Further studies are necessary to address this.

It is important to mention that a negative temporal artery biopsy does not rule out the diagnosis of temporal artery GCA, since the changes can be patchy with skip areas of uninvolved artery. Although serial sectioning of the temporal artery biopsy is recommended, not all cases will be diagnosed histologically[33]. Actually, after review of the medical records of five of our biopsy-negative HPV-positive cases, in one case, the patient showed dramatic improvement after treatment with steroids (possible false negative case which was not included as a HPV-positive case in our statistical analysis).

It is also worth noting that of the 16 histologically-proven GCA cases that had HPV, 11 were high risk genotype 16. Only five cases were positive for low risk HPV genotype 6. Although, the importance of distinguishing low risk from high risk HPV genotypes has been established in the pathogenesis of neoplasms, what its significance might be in inflammatory disorders such as GCA is currently unknown.

In summary, we have identified HPV DNA in the majority of histologically-proven giant cell (temporal) arteritis specimens and validated these results by a completely independent outside laboratory assay. The association raises questions regarding the biology of HPV infections, when dissemination occurs, and what this dissemination means clinically. Such studies are required to understand the significance of the association in our series.