Detection Rate of Human Papilloma Virus Genotype 16 / 18 infections in Brain Tissues from a group of Iraqi Patients with Glioblastoma Multiforme: An In Situ Hybridization Study.

Introduction

Gliomas are the most prevalent and malignant type of primary central nervous system tumors. Gliomas are classified according to their cellular origin into: oligodendrogliomas, ependymomas, mixed gliomas and the astrocytic tumors (astrocytomas, 4 grades: I to IV), where among the latter group, Glioblastoma Multiforme which is a rare and aggressive form of brain cancer classified as a grade IV astrocytoma. Glioblastoma multiforme (GBM) is the most prevalent and deadly type of primary brain tumor in humans (has an extremely poor survival rate owing to its aggressive and malignant nature). According to cancer 2010 statistics, GBM makes up the majority (>60%) of all primary brain tumors, while according to Central Brain Tumor Registry of US 2008–2012 data makes for 45% of these tumors.^1–11

The etiology of central nervous system CNS tumors remains debatable; however, over the last decade, there is growing evidence and consensus have been proposed regarding the hypothesis that certain human viruses may be associated with and/or implicated in these central nervous system (CNS) tumors.^12,13 Previous scientific studies have highlighted the involvement of viral infections in the development and progression of glioblastoma tumors. In the last decade, researchers have suggested a role for viral agents, such as Human CMV and JC virus, in the process of gliomagenesis,¹⁴ while Zavala-Vega et al. research in 2019¹⁵ identified the presence of Epstein-Barr Virus in glioblastoma cases.

Human papillomavirus (HPV) is a DNA virus with a genome of approximately 8000 base pairs belonging to the Papillomaviridae family. Over the last two decades, more than 450 genotypes of human papilloma viruses were recognized to have the ability to infect human mucosal surfaces and cutaneous tissues.¹⁶

At least 25 recognized HPV genotypes have been identified to cause cancers affecting different anatomical sites in the body, while the remaining HPV genotypes are considered to have a low oncogenic risk causing non-cancerous human growth and lesions and are associated with benign warts and papillomas. Several studies have demonstrated that in patients with persistent genital infection with HPV16 and HPV18 and their involvement in precancerous cervical lesions are susceptible in 82% to have cervical cancer after 15-20 years.^17–22 In addition, an increased detection rate of HPV infections in head and neck cancers has been reported in the last few years, and most have been documented in oropharyngeal cancers.²³

Human papillomavirus (HPV) is considered the most commonly recognized oncogenic virus in humans. The present study aimed to evaluate the occurrence rates of HPV genotypes 16 and 18 infection in resected brain tissue samples obtained from patients who sustained operations for glioblastoma multiforme [GBM] (astrocytoma grade IV) and to compare it with non-tumorous brain tissues as their control counterparts.

Materials and methods

This study was performed in accordance with the Declaration of Helsinki (https://www.wma.net/policies-post/wma-declaration-of-helsinki). Ethical approval was obtained from the College of Medicine- University of Baghdad Deanship No. 132 (25/5/2025). Verbal consent has been obtained from all individuals or from a suitable legal guardian because of limited time right before admission for surgery. In this retrospective case-control study, a total number of 74 tissue specimens were obtained from patients who had sustained operations in neurosurgical theatres for glioblastoma multiforme. Those patients were aged between 4 and 78 years, while the other 29 tissue specimens that were obtained and enrolled from control counterpart patients, aged 23 to 72 years, and had been operated in these theatres for many other non-tumorous neurosurgical diagnoses, where these tissues on histopathological examination have diagnosed not in favor of benign tumors and cancers in the CNS, were enrolled as a control group for this study. The criteria established by the WHO Health Organization were used for tumor categorization. The institutional review board and local ethics committee permission were obtained from the project.

Results

CISH detection rates of HPV 16/18 DNA in the studied GBM and control groups

CISH results of HPV 16/18 -DNA according to their signal scoring and intensity

Table 3 shows the positive -CISH detection results of HPV 16/18 where 31.1% (23 out of 74 cases) from GBM group showed positive signals, including 47.8% were of weakly stained (score I) followed by 30.4% and 21.8 % (had moderate and high scores (score II & III), respectively. The statistical analysis of positive HPV 16/18 -CISH score results showed a highly significant difference (P < 0.001) depending on (Chi-square & Phi tests).

Table 3. CISH results of HPV 16/18 –DNA in GBM and control groups according to the signal scoring and intensity.

CISH results of HPV 16/18-DNA		Control patients with non-tumorous brain pathologies (n = 29)		Brain Glioblastoma Multiforme (GBM) patients (N = 74)		P value
		N	%	N	%
Negative-CISH RESULTS		28	96.5	51	68.9	P < 0.001
Positive-CISH RESULTS		1	3.5	23	31.1
SCORING	I	1	100	11	47.8
	II	0	0.0	7	30.4
	III	0	0.0	5	21.8
INTENSITY	I	1	100	14	60.9	P = 0.004
	II	0	0.0	7	30.4
	III	0	0.0	2	8.7
Mean Rank		95.00		85.7

In addition, Table 3 reveals 60.9% of GBM tissues had intensity (I), followed by 30.4% with intensity (II), and 8.7% with intensity (III) ( Figures 1 & 2). Statistically significant differences were also observed between the negative, score I, II, and III intensities of CISH reactions at the 5 percent level (P = 0.004) in the GBM tissue group.

Figure 1. Positive -CISH reaction detection of HPV 16/18-DNA with a low score and moderate intensity (40X).

Figure 2. Positive HPV 16/18-DNA-CISH reaction in Glioblastoma Multiforme tissues with moderate score grading and moderate intensity (40X).

Scoring assessment results of CISH reaction for HPV 16/18-DNA detection in brain Glioblastoma Multiforme tissues. The detection results of CISH-DNA for HPV 16/18 via staining with BCIP/NBT (stained blue), while the cellular nuclei were stained red by the counterstain Nuclear Fast Red.

The physical state of CISH signaling reactions for HPV 16/18 -DNA detection according to their nuclear localization

The physical states of HPV 16/18 -DNA detection in the studied Glioblastoma Multiforme tissue group have revealed an episomal physical state in (56.5%), whereas (43.5%) have showed an integrated state ( Table 4).

Table 4. Integrated and episomal physical state forms of HPV 16/18 -DNA among studied tissues from Glioblastoma Multiforme group.

State forms of HPV 16/18 -DNA	Glioblastoma Multiforme (n=23)
Episomal State	13 (56.5%)
Integrated State	10 (43.5%)

The CISH-results of HPV 16/18 -DNA detection in GBM group of tissues according age strata and gender of the patients

The brain tissues related to GBM patients in the age stratum (41-60 years) have revealed HPV 16/18 -DNA detection in 10.8%, while in the age stratum (4-20 years), (21-40 years), and (61-78 years) the brain tumor tissues have revealed HPV 16/18 -DNA detection in 5.4%, 6.8%, and 8.1%, respectively. Significant differences (P<0.05) were found when HPV 16/18 -DNA detection rates in these age groups were compared ( Table 5).

Table 5. Frequency of positive-HPV 16/18 CISH reaction results in brain tumors tissues from GBM patients according their age Strata.

	Years	HPV 16/18 –DNA CISH results in GBM tissues			P value
		No.	Positive	Negative
Age stratum	4-20	15	4	11	chi square test P = 0.03
		20.3%	5.4%	14.9%
	21-40	18	5	13
		24.3%	6.8%	17.6%
	41-60	20	8	12
		27.0%	10.8%	16.2%
	61-78	21	6	15
		28.4%	8.1%	20.3%
Total		74	23	51
		100%	52.1%	47.9%

Table 6. Positive percentages of HPV 16/18 -CISH results in GBM patients based on their gender.

GBM Patients Tissues	HPV 16/18 –DNA CISH results
	Positive	%
Males (N = 43)	15	65.2
Females (N = 31)	8	34.8
The analysis Statistical	P = 0.03

Discussion

Glioma is the most common type of primary brain tumor arising from the carcinogenesis of glial cells in the brain and spinal cord. The World Health Organization (WHO) has provided a classification for malignancy levels of gliomas and categorizes gliomas into four different grades based on their histopathological characteristics: Grade I gliomas are lesions with low proliferative potential, with the higher grade II-IV gliomas being undifferentiated, highly malignant, invasive, and especially GBM, classified as a Grade IV glioma, being the most malignant and difficult to treat and deadly type of brain cancers that have an extremely poor prognosis. Many underlying mechanisms driving glioma tumorigenesis remain unknown; however, studying the molecular mechanisms driving GBM is key to advancing diagnostic and treatment approaches.^12,26–28

World-Globocan 2020 has announced that the global GBM incidence of brain cancers has increased from 2009 to 2020, from less than 10 per 100,000 to 10.74 per 100 000.^29,30 According to the registration by IARC-Globocan-Iraq -2020, an incidence of brain cancers of 10.18 per 100 000 has been reported in Iraq.³¹

In accordance with the WHO histopathological grading system for gliomas,^2–6 this study analyzed tissue samples from patients with the most malignant form of astrocytic brain tumor, glioblastomas (grade IV).

In the current study, informed consent was obtained from each patient included in the study, we assessed the total number of archived, paraffin-embedded tumor tissue biopsies were obtained from (74) patients aged 4 to 73 years, comprised of 58.1% males and 41.9.% female patients with primary glioblastoma multiforme (GBM) as well as another number control of brain tissues whom were obtained from²⁹ patients, aged 23 to 72 years, comprised of 62.1% male and 37.9% female patients, and had been operated in these theatres for many other CNS diseases lacking tumor pathology (whether benign and cancers) enrolled as a control group of this study ( Table 1).

Previous reports have suggested a potential association of several types of neurotropic viruses, frequently causing both acute and chronic viral CNS infections, with CNS tumors,¹³ which enter the CNS through blood circulation (viremia) or by crossing the blood-brain barrier (BBB) and via peripheral nerve endings.^32,33 During the past two decades, many studies have reported that persistent high-risk HPV infections, including HPV16 and 18 genotypes, are associated with the majority of cervical cancers, as well as increased HPV rates in head and neck cancers and increased numbers of oropharyngeal SCCs.^34–37

This study, and up to our best knowledge, represents the first study, at least in Iraq. This study aimed to determine the genotype 16 and 18 rates of Human Papilloma Virus in glioblastoma multiforme patients who underwent operations in the neurosurgical theatres at the Specialized Surgeries Hospital/Baghdad, using chromogenic in situ hybridization (CISH) for DNA localization of genotypes 16 and 18 in formalin-fixed paraffin-embedded resected glioblastoma multiforme tissues.

In the current study, the results of detection of genotypes 16 and 18 and according to the statistical analysis of the positive CISH- reactions have showed highly significant differences (P < 0.001) between the astrocytoma grade 4 (glioblastoma multiforme) cases, which revealed 31.1% (23 out of 74 cases) positivity for genotypes 16 and 18 when were compared to its control group (other pathological brain cases that have non-tumorous tissues that have revealed 3.5% (1 out of 29 cases)) positive in situ hybridization (ISH reactions) ( Table 3).

Arsene et al. (2022)¹³ reported HPV DNA in 25% of meningiomas, 25.86% of gliomas, and 7.5% of controls. HPV was significantly more frequent in glioma patients than in controls, with the glioblastoma subgroup showing a higher positivity rate (29.5%) compared to controls.

HPV type-dependent features have been shown among cancer histological types,³⁸ where most HPV 16-positive cancers are squamous cell carcinomas, whereas approximately 50% of HPV 18-positive cancers are adenocarcinomas.³⁹ Furthermore, HPV 18 is more likely to integrate the viral genome into the host genome than HPV 16.⁴⁰ Our findings on HPV in glioblastoma align with Vidone et al.,¹⁴ Adnan's al.,⁴¹ and Hashida's al.,⁴² who reported 25–28% HPV positivity in glioblastoma patients, suggesting HPV's potential role in tumor pathophysiology. Limam et al.⁴³ were recently observed an even higher HPV positivity rate of 39.3% in glioblastoma cases. In this respect, Adnan et al.⁴¹ reported that the majority of HPV-positive patients with Primary Glioblastoma in Pakistan⁴¹ harbored both HPV 16 and 18 types, of which 16% were HPV-type 16 and 20% were HPV-type 18.

The evaluation trials of HPV prevalence in patients with different types of brain and CNS tumors are crucial to delineate where and when such HPV infections in these CNS tissues have been acquired and for how long these viruses are latent in them. Moreover, the use of in situ hybridization technique is valid for disclosing the histopathological examination of the physical states of HPV-DNA in cellular chromosomal DNA. The researchers frequently observed integration of this virus in the majority of HPV-positive cervical cancers, and a variable percentage of HPV-positive oropharyngeal SCC harbored integrated HPV; however, the identification of integration is influenced by the type of detection method. Furthermore, the histopathological ratings of HPV-DNA in relation to the grading of astrocytomas reaching glioblastomas are also important to delineate their roles in CNS tumorigenesis, whether at early or late events.^44–47

Regarding either HPV physical states of HPV 16/18 DNA CISH- detection signals; both expressed forms (the integrated and episomal physical forms of HPV 16/18 DNA) were seen in the currently researched glioblastomas tissues. Importantly, the glioblastoma tissues have revealed an integrated physical pattern in 43.5% (10 out of 23 tissues) of the total positive CISH test signals of HPV genotype 16/18 in this group of tissues, whereas episomal physical state was revealed in (56.5%) ( Table 4).

Researchers have found that such viral integration could contribute to oncogenesis via deregulation of HPV E6 and E7 oncogene expression, which inactivates p53 and pRB genes, and that these factors increase cell proliferation and genetic instability.^48–50

Diagnosing and treating CNS infections is urgent and challenging because few effective antiviral drugs are currently available. Currently, several studies have shown the efficacy of the Human Papillomavirus (HPV) vaccine in successfully preventing HPV 16/18 infections and reducing the risk of cervical cancer and the development of other HPV-related cancers.⁵¹

In conclusion, our results suggest that the current detection rates of HPV 16/18 infection in glioblastoma multiforme (grade 4) tissues seem to be significantly associated with these primary tumors of the CNS and could shed light on the possible roles of such important high-risk viral infections in the pathogenesis and/or carcinogenesis of these currently studied glioblastoma multiforme as well as astrocytoma grade 2 tissues from this group of Iraqi patients.

On the other hand, it can also be concluded that the current findings of HPV 16/18 DNA detection in glioblastoma tissues, and in part, also indicate that these tissues might represent a possible reservoir site for spreading such important high oncogenic risk Human Papilloma infection to other tissue localizations, either to the neighboring tissues and/or at distant anatomical sites in these patients. In addition, the fate of such HPV 16/18 infections in these tumors could either play part in the future malignant pathogenesis of these lesions, or are just transiently existing HPV 16/18 infections that will be managed later efficiently by the immune system of those infected patients.