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.

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Zeiny" }, { "@type": "Person", "name": "Ali K. Al-Shalchy" }, { "@type": "Person", "name": "Saad Hasan Mohammed Ali" }, { "@type": "Person", "name": "Shakir H. Mohammed Al-Alwany" }, { "@type": "Person", "name": "Athraa Y. Al-Hijazi" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " Background Over 450 human papillomavirus (HPV) genotypes have been identified, with high-risk HPV-16 and -18 linked to head, neck, and central nervous system (CNS) cancers, including glioblastoma multiforme (GBM), a highly aggressive tumor with poor prognosis. Objective This study assessed HPV-16/18 DNA detection rates via in situ hybridization (ISH) in GBM tissues from patients in Baghdad neurosurgical wards, compared to non-malignant CNS controls. Materials and Methods Seventy-four archived GBM tissues (ages 4–73) and 29 benign/non-cancerous CNS controls (ages 23–72) were analyzed using ISH with genotype-specific DNA probes. Results HPV-16/18 DNA was detected in 23/74 (31.1%) GBM tissues versus 1/29 (3.5%) controls. Among positive GBM cases, detection scores were weak (47.8%), moderate (30.4%), or high (21.8%), while intensity scores were high (60.9%), moderate (30.4%), or weak (8.7%). The single positive control exhibited low detection/intensity. Conclusions The significantly higher HPV-16/18 prevalence in GBM tissues suggests a potential role in carcinogenesis and gliosis pathogenesis, highlighting the oncogenic risk of HPV in this subset of glioblastomas. 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F1000Research 2025, 14 :869 ( https://doi.org/10.12688/f1000research.166556.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article 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. [version 1; peer review: 2 approved with reservations] Sarmad M.H. Zeiny https://orcid.org/0000-0002-1976-9157 1 , Ali K. Al-Shalchy 2 , Saad Hasan Mohammed Ali 3 , Shakir H. Mohammed Al-Alwany 4 , Athraa Y. Al-Hijazi 5 Sarmad M.H. Zeiny https://orcid.org/0000-0002-1976-9157 1 , Ali K. Al-Shalchy 2 , [...] Saad Hasan Mohammed Ali 3 , Shakir H. Mohammed Al-Alwany 4 , Athraa Y. Al-Hijazi 5 PUBLISHED 03 Sep 2025 Author details Author details 1 Medical Microbiology & Immunology Department, University of Baghdad, College of Medicine, Baghdad, Baghdad Governorate, 0000, Iraq 2 Surgery Medical City complex / Chief of the Scientific council of Neurosurgery in the Iraqi Board, University of Baghdad, College of Medicine, Baghdad, Baghdad Governorate, 0000, Iraq 3 Medical Virology, Al Mustaqbal Universirty, Babylon, 0000, Iraq 4 Professor in Medical Virology, College of Science, nv=, University of Babylon, Babylon, Iraq 5 Histopathology, Al-Mustaqbal University, Babylon, Iraq Sarmad M.H. Zeiny Roles: Software, Supervision, Writing – Review & Editing Ali K. Al-Shalchy Roles: Conceptualization, Investigation, Methodology Saad Hasan Mohammed Ali Roles: Formal Analysis, Validation, Writing – Original Draft Preparation Shakir H. Mohammed Al-Alwany Roles: Investigation, Project Administration, Software, Supervision, Validation Athraa Y. Al-Hijazi Roles: Data Curation, Formal Analysis, Investigation, Resources OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Genomics and Genetics gateway. This article is included in the Oncology gateway. This article is included in the Cell & Molecular Biology gateway. This article is included in the Glioblastoma Heterogeneity collection. Abstract Background Over 450 human papillomavirus (HPV) genotypes have been identified, with high-risk HPV-16 and -18 linked to head, neck, and central nervous system (CNS) cancers, including glioblastoma multiforme (GBM), a highly aggressive tumor with poor prognosis. Objective This study assessed HPV-16/18 DNA detection rates via in situ hybridization (ISH) in GBM tissues from patients in Baghdad neurosurgical wards, compared to non-malignant CNS controls. Materials and Methods Seventy-four archived GBM tissues (ages 4–73) and 29 benign/non-cancerous CNS controls (ages 23–72) were analyzed using ISH with genotype-specific DNA probes. Results HPV-16/18 DNA was detected in 23/74 (31.1%) GBM tissues versus 1/29 (3.5%) controls. Among positive GBM cases, detection scores were weak (47.8%), moderate (30.4%), or high (21.8%), while intensity scores were high (60.9%), moderate (30.4%), or weak (8.7%). The single positive control exhibited low detection/intensity. Conclusions The significantly higher HPV-16/18 prevalence in GBM tissues suggests a potential role in carcinogenesis and gliosis pathogenesis, highlighting the oncogenic risk of HPV in this subset of glioblastomas. READ ALL READ LESS Keywords Glioblastoma multiforme (grade IV astrocytoma,); Human Papillomavirus; HPV 16; HPV 18; In Situ Hybridization ; ISH. Corresponding Author(s) Sarmad M.H. Zeiny ( [email protected] ) Close Corresponding author: Sarmad M.H. Zeiny Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2025 Zeiny SMH et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Zeiny SMH, Al-Shalchy AK, Hasan Mohammed Ali S et al. 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. [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :869 ( https://doi.org/10.12688/f1000research.166556.1 ) First published: 03 Sep 2025, 14 :869 ( https://doi.org/10.12688/f1000research.166556.1 ) Latest published: 03 Sep 2025, 14 :869 ( https://doi.org/10.12688/f1000research.166556.1 ) 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 2019 15 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. Tissue specimens The specimens were collected during the period (19/1/2022–20/4/2025) from the archives of Histopathology department at Specialized Surgeries Hospital in Baghdad and two major Iraqi teaching hospitals in Babylon and AL-Najaf governorates as well as many private laboratories in these governorates. The main preparatory steps for paraffin-embedded tissue sectioning and histopathological processing, as well as all sequential steps for achieving HPV16/18-DNA detection by chromogenic in-situ hybridization (CISH) procedure and all CISH validation and scoring assessments were performed at the Histopathology Laboratory at the College of Dentistry/Al- Mustaqbal University, Babylon, Iraq, and in the Virology Laboratory of Molecular Science Division at the College of Science/University of Babylon, Babylon, Iraq. Detection of HPV 16/18 DNA by CISH The method used in this study was performed on tissue sections of 4-μm thickness, which were specified and examined for the detection of HPV 16/18-DNA via the use of a recent version of chromogenic in situ hybridization (CISH) kit (Zyto Vision, GmbH, Bremerhaven, Germany) that utilized a specifically designed oligonucleotide DNA probe (labeled by digoxigenin) and specifically targeted the DNA of the HPV 16 and 18 genotypes. The slides were incubated for 1 h at 70°C, followed by immersion twice in absolute xylene for 5 min, rehydration in sequential steps at room temperature, and finally subjected to a drying step at 37°C for 5 min, followed by the application of pepsin solution (for 45 min at 37°C) and rinsing in distilled water, followed by air drying. The main CISH steps started by adding the specific oligonucleotide probe and denaturation at 75°C for 5 min, followed by hybridization at 37°C for 18 h and washing at 55°C for 5 min in 1 × TBS wash buffer. Then, anti-digoxigenin-conjugated alkaline phosphatase was applied (incubated for 30 min at 37°C), washed once in TBS buffer and twice in distilled water for 5 min, and then rinsed in detergent buffer (5 min) and BCIP/NBT (30 min at 37°C). The slides were counterstained with Nuclear Fast Red, sequentially dehydrated with ethyl alcohol, and mounted with a Disteren Plasticizer Xylene. 24 Analysis of the CISH color signaling results Positive nuclear CISH staining was determined in 10 high-power fields that elicited a blue color in the examined cells, representing the complementary sites of the probes to their specific HPV 16/18-DNA. The positive patterns of CISH signals were classified as follows: 1. Diffuse (D): Nuclei that were completely stained. 2. Punctuated (P): Representative nuclei showing distinct dot-like signals. 3. Mixed = Diffuse + Punctuated (D/P): The examined cells with their nuclei showing both positive patterns of CISH signals. 25 To quantify the in situ hybridization signals, light microscopy was used to count the positive cells at X400, giving the intensity of the positive signal of the reactions (as low, moderate, and high) and percentage score results based on the number of cells with positive signals. The scores of the average of positive cells that were counted in ten different fields of 100 cells for each sample were determined and assigned to one of the following three percentage score categories: Score (1) = 1-25%, Score (2) = 26-50%, Score (3) > 50%. 24 Statistical analysis A chi-squared test for calculating the statistical significance of parameters was performed, and using the SPSS-23 package, an analysis of the relationship between variables was performed. A p-value equal to or lower than 0.05 indicates a significant relationship. Results Some demographic factors in relation to the pathological description of the studied groups Age distribution of studied groups The age of 74 patients with glioblastoma multiforme ranged from 4 to 78 years with a mean age of 47 (S.D 11.9) years, while compared to the age of 29 control counterpart patients with non-tumorous brain pathologies who had ranged from 23 to 72 years with a mean age of 44 (S.D 12.8) years, no significant differences were detected between the mean age of these 2 groups (> P 0.05) ( Table 1 ). Table 1. The age distribution of the researched GBM patients group. Study group No. Mean age (years) S.D S.E Minimum Maximum Brain Glioblastoma Multiforme Patients 74 47 11.9 1.7 4 78 Control Patients with Non-Tumorous Brain Pathologies 29 44 12.8 2.3 23 72 Statistical Analysis Non-significant (P > 0.05) = 0.06 Gender distribution of studied GBM group Regarding the patients who suffering from GBM, the percentage/ number of males was higher (58.1%:43) than the percentage/number of females (41.9%:31). While, it was found that the percentage/number of males in control patients group was (62.1%:18), while the remaining 11 cases (37.9%) were females. The male/female ratios for the GBM and control patients were 1.38:1 and 1.63:1, respectively ( Table 2 ). A non-significant difference was found between patients in the GBM and control groups according to their gender ratio (P = 0.05). Table 2. Results of gender distribution of the studied GBM and control groups. Variable GBM patients N (%) Control patients N (%) P Sex Male 43 (58.1%) 18 (62.1%) 0.05 Female 31 (41.9%) 11 (37.9%) 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 Spearman's Rho statistical testing to evaluate the studied markers (Grade, sex, age and HPV 16/18 -CISH) in brain tissues from GBM Patients A strong positive relationship (with a significant correlation) was found between HPV 16/18 and Grade IV (GBM) tumors patients (r = 0.347, P = 0.04). Moreover, there was a significant correlation between HPV 16/18 according to age of patients with brain GBM tumors (r = 0.397, P = 0.04). However, there was no significant correlation between GBM tumors and the gender of the patients with brain GBM tumors (r = 0.793, P = 0.05). In addition, a non-significant correlation was found between sex and age of the patients (r = 0.756, P = 0.06) ( Table 7 ). Table 7. Spearman’s Rho statistical testing of age, sex, grade, and HPV 16/18 -CISH to evaluate the studied markers in brain tumors. Spearman’s rho Patient’s age Patient’s sex GBM tumors HPV 16/18 HPV 16/18 r 0.379 0.347 P 0.03 * 0.04 * GBM tumors r 0.756 P 0.05 * Sex r 0.793 p 0.05 * Age r 0.397 P 0.04 * * Correlation is highly significant (P < 0.05). 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 from 29 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 Pakistan 41 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. Data availability 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. [Sarmad M. H. Zeiny] Data are available at zenodo ([]( https://doi.org/10.5281/zenodo.15634620 ). 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Oncol. 2024; 42 : 10507. Publisher Full Text 52. Zeiny S: 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. Zenodo. 2025. Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 03 Sep 2025 ADD YOUR COMMENT Comment Author details Author details 1 Medical Microbiology & Immunology Department, University of Baghdad, College of Medicine, Baghdad, Baghdad Governorate, 0000, Iraq 2 Surgery Medical City complex / Chief of the Scientific council of Neurosurgery in the Iraqi Board, University of Baghdad, College of Medicine, Baghdad, Baghdad Governorate, 0000, Iraq 3 Medical Virology, Al Mustaqbal Universirty, Babylon, 0000, Iraq 4 Professor in Medical Virology, College of Science, nv=, University of Babylon, Babylon, Iraq 5 Histopathology, Al-Mustaqbal University, Babylon, Iraq Sarmad M.H. Zeiny Roles: Software, Supervision, Writing – Review & Editing Ali K. Al-Shalchy Roles: Conceptualization, Investigation, Methodology Saad Hasan Mohammed Ali Roles: Formal Analysis, Validation, Writing – Original Draft Preparation Shakir H. Mohammed Al-Alwany Roles: Investigation, Project Administration, Software, Supervision, Validation Athraa Y. Al-Hijazi Roles: Data Curation, Formal Analysis, Investigation, Resources Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (1) version 1 Published: 03 Sep 2025, 14:869 https://doi.org/10.12688/f1000research.166556.1 Copyright © 2025 Zeiny SMH et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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F1000Research 2025, 14 :869 ( https://doi.org/10.5256/f1000research.183561.r421158 ) The direct URL for this report is: https://f1000research.com/articles/14-869/v1#referee-response-421158 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 07 Nov 2025 Lucia E. Azuara-Alvarez , Facultad de Ciencias de la Salud, Autonomous University of Baja California, Tijuana, Mexico, Tijuana, Baja California, Mexico Giovanni Palomino Vizcaino , Facultad de Ciencias de la Salud, Autonomous University of Baja California, Tijuana, Baja California, Mexico Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.183561.r421158 This Authors study examined the presence of high-risk human papillomavirus (HPV) genotypes 16 and 18 in brain tissues from Iraqi patients with glioblastoma multiforme (GBM), revealing that 31.1% of GBM samples tested positive compared to only 3.5% of non-tumorous controls. ... Continue reading READ ALL This Authors study examined the presence of high-risk human papillomavirus (HPV) genotypes 16 and 18 in brain tissues from Iraqi patients with glioblastoma multiforme (GBM), revealing that 31.1% of GBM samples tested positive compared to only 3.5% of non-tumorous controls. Using chromogenic in situ hybridization (CISH), researchers found both episomal and integrated viral DNA forms, with significant associations between HPV presence and male sex. They suggest a potential oncogenic role of HPV in GBM pathogenesis, aligning with previous international reports and highlighting the need for further investigation into viral contributions to central nervous system tumors. We approved With Reservations The introduction provides a general overview of glioma classification and prevalence, and briefly mentions the potential involvement of viral infections in central nervous system (CNS) tumors. However, to enhance its scientific depth and relevance, several key improvements are recommended. While the authors reference viruses such as CMV, JC virus, and Epstein-Barr virus in gliomagenesis, the specific relationship between human papillomavirus (HPV) and glioblastoma multiforme (GBM) is not adequately developed. The introduction would benefit from a more focused discussion of prior studies that have investigated HPV presence in brain tumors, particularly glioblastomas, and from articulating the rationale for exploring this association. The molecular mechanisms by which HPV exerts its oncogenic effects are notably absent. A concise explanation of how HPV oncoproteins E6 and E7 disrupt tumor suppressor pathways—specifically through inactivation of p53 and pRB—would provide essential context. This would help justify the relevance of detecting HPV DNA in GBM tissues and clarify the biological plausibility of its involvement in CNS tumorigenesis. The introduction should more clearly define the knowledge gap this study aims to address. Emphasizing the limited evidence on HPV's role in brain tumors and the need for further investigation would strengthen the framing of the research question and increase the impact of the study. To strengthen the validity and interpretability of the findings, we recommend that the authors include both positive and negative controls in their experimental design and results section. The absence of clearly defined control benchmarks limits the ability to assess the specificity, sensitivity, and reliability of the CISH detection method used for HPV 16/18. Incorporating well-characterized control samples would help clarify the criteria applied for signal classification, intensity grading, and physical state determination, thereby enhancing the rigor of the analysis and supporting the reproducibility of the study. To enhance the robustness of their findings, the authors are encouraged to include an assessment of HPV L1 capsid protein as an indirect means of corroborating the viral presence detected by CISH. The detection of L1 would help confirm active or latent HPV infection and provide additional support for the specificity of the hybridization signals. Furthermore, evaluating p53 protein in the same tissue sections would be highly valuable, as it could offer functional evidence of E6 oncoprotein activity. Given that E6 is known to mediate p53 degradation, observing altered or reduced p53 in HPV-positive GBM tissues would strengthen the argument for a mechanistic link between HPV infection and gliomagenesis. The viral gene expression program of human papillomavirus (HPV) is tightly regulated by the stratification and differentiation of epithelial tissues, particularly in the cervical epithelium where its transcriptional activity is well-characterized. Given this context, the manuscript presents a significant gap by not providing evidence of viral protein expression in glioblastoma tissues, which is essential to support any proposed association between HPV infection and GBM pathogenesis. To strengthen the argument, the authors should either adjust the wording to reflect the limitations of their findings or include immunohistochemical or molecular data demonstrating the presence of viral proteins such as E6 or E7. Furthermore, the assumption that HPV proteins are produced in neuronal or glial cells requires careful justification, as the virus’s expression is known to be highly dependent on epithelial-specific regulatory mechanisms. A discussion addressing how HPV gene expression could occur in non-epithelial tissues like the brain is necessary to clarify the biological plausibility of the proposed link. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Partly References 1. Palomino-Vizcaino G, Bañuelos-Villegas E, Alvarez-Salas L: The Natural History of Cervical Cancer and the Case for MicroRNAs: Is Human Papillomavirus Infection the Whole Story?. International Journal of Molecular Sciences . 2024; 25 (23). Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: molecular diagnostics, aptamer engineering, and nanobiotechnology for viral detection and inhibition, particularly HPV. We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Azuara-Alvarez LE and Vizcaino GP. Reviewer Report For: 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. [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :869 ( https://doi.org/10.5256/f1000research.183561.r421158 ) The direct URL for this report is: https://f1000research.com/articles/14-869/v1#referee-response-421158 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Piranlioglu R and Runco E. Reviewer Report For: 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. [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :869 ( https://doi.org/10.5256/f1000research.183561.r418824 ) The direct URL for this report is: https://f1000research.com/articles/14-869/v1#referee-response-418824 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Oct 2025 Raziye Piranlioglu , Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA Emerenziana Runco , Brigham and Women’s Hospital, Harvard Medical School, Massachusetts, Boston, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.183561.r418824 Comments to Authors Zeiny et al. discusses the potential involvement of high-risk human papillomavirus (HPV) types 16 and 18 in glioblastoma multiforme (GBM) in Iraqi patients using chromogenic in situ hybridization (CISH). The authors examined 45 GBM tissue samples ... Continue reading READ ALL Comments to Authors Zeiny et al. discusses the potential involvement of high-risk human papillomavirus (HPV) types 16 and 18 in glioblastoma multiforme (GBM) in Iraqi patients using chromogenic in situ hybridization (CISH). The authors examined 45 GBM tissue samples and 30 non-neoplastic brain controls and detected HPV 16/18 in 31.1% of GBM and 3.5% of controls. They conclude that infection with HPV may play a role in GBM pathogenesis. We are in favor of indexing; however, this is contingent upon the authors addressing the revisions and concerns outlined below. Major Comments 1. Introduction gives a general overview of glioma and HPV biology but should be improved, setting the focus on more prevalence of HPV infection in Iragi population. Also, the authors should revise their classification of gliomas to align with the most recent updates, as their current description reflects the pre-2016 WHO classification, which was based solely on histogenesis (i.e., the tumor's cellular origin). While this historical approach isn't incorrect, it is now considered outdated in contemporary scientific literature. Additionally, the authors should replace the outdated 2010 prevalence statistics for glioblastoma (GBM) with more current data, as updated figures are now available. Please refer to the sources below: https://seer.cancer.gov/statfacts/html/brain.html https://pubmed.ncbi.nlm.nih.gov/36006639/ Berger et al., (Reference 1) 2. The CISH method is clearly outlined; however, its reproducibility relies heavily on the inclusion of both positive and negative controls. The criteria for interpreting positive signals should be clarified, as distinguishing true positives from false positives is currently challenging. To improve clarity, the authors should present staining results from control groups. A side-by-side comparison showing low, moderate, high, and negative signals from these controls would be valuable for distinguishing signal intensities more effectively. Moreover, the study would be enhanced by a brief description of sample handling (fixation, storage time) and DNA quality control, as paraffin-embedded material can affect viral DNA detection. 3. Determining if the HPV infection is ongoing or latent will enhance impact of study. Since CISH detects viral DNA but cannot distinguish between active and latent infections, the use of qPCR—or RT-PCR—would be helpful to assess viral activity and strengthen the interpretation of results. 4. Does the presence of HPV influence patient survival outcomes or treatment response? Clarifying whether HPV status has any prognostic or predictive value would enhance the clinical relevance of the findings. 5. One important variable not included in this study is the HPV vaccine status of the patients. Since HPV vaccination (for genotypes 16/18) significantly reduces infection rates, information regarding whether GBM and control subjects were vaccinated could be crucial. If these data are available, the authors must analyze whether vaccination is linked to the prevalence of HPV detection. If unavailable, this limitation must be explicitly declared and accounted for as a confounder. Future studies would be enhanced by stratifying patients by vaccination status to ascertain whether detected HPV DNA is attributable to actual infection as opposed to residual viral material not actually involved in disease pathogenesis. 6. The paper appropriately cites several studies but, in most instances, it confuses association with causation. The language must be more precise to avoid the suggestion of a causal link between HPV and GBM. 7. The authors must expand the limitations section: small sample size, retrospective study, paraffin material used, and no molecular confirmation (PCR or RNA-ISH). 8. Have the authors investigated whether there are any genetic differences in the GBM tissues, such as mutations or amplifications of specific genes (e.g., P53 ) that could influence susceptibility to HPV infection? Exploring such molecular alterations may provide important context for understanding the interaction between HPV and tumor biology. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Partly References 1. Berger T, Wen P, Lang-Orsini M, Chukwueke U: World Health Organization 2021 Classification of Central Nervous System Tumors and Implications for Therapy for Adult-Type Gliomas. JAMA Oncology . 2022; 8 (10). Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Cancer Immunotherapy, Brain Tumor Immunology, Oncolytic Virotherapies in Glioblastoma. We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Piranlioglu R and Runco E. Reviewer Report For: 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. [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :869 ( https://doi.org/10.5256/f1000research.183561.r418824 ) The direct URL for this report is: https://f1000research.com/articles/14-869/v1#referee-response-418824 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 03 Sep 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 1 03 Sep 25 read read Raziye Piranlioglu , Brigham and Women’s Hospital, Harvard Medical School, Boston, USA Emerenziana Runco , Brigham and Women’s Hospital, Harvard Medical School, Massachusetts, USA Lucia E. Azuara-Alvarez , Autonomous University of Baja California, Tijuana, Mexico, Tijuana, Mexico Giovanni Palomino Vizcaino , Autonomous University of Baja California, Tijuana, Mexico Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Vizcaino G et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 07 Nov 2025 | for Version 1 Lucia E. Azuara-Alvarez , Facultad de Ciencias de la Salud, Autonomous University of Baja California, Tijuana, Mexico, Tijuana, Baja California, Mexico Giovanni Palomino Vizcaino , Facultad de Ciencias de la Salud, Autonomous University of Baja California, Tijuana, Baja California, Mexico 0 Views copyright © 2025 Vizcaino G et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This Authors study examined the presence of high-risk human papillomavirus (HPV) genotypes 16 and 18 in brain tissues from Iraqi patients with glioblastoma multiforme (GBM), revealing that 31.1% of GBM samples tested positive compared to only 3.5% of non-tumorous controls. Using chromogenic in situ hybridization (CISH), researchers found both episomal and integrated viral DNA forms, with significant associations between HPV presence and male sex. They suggest a potential oncogenic role of HPV in GBM pathogenesis, aligning with previous international reports and highlighting the need for further investigation into viral contributions to central nervous system tumors. We approved With Reservations The introduction provides a general overview of glioma classification and prevalence, and briefly mentions the potential involvement of viral infections in central nervous system (CNS) tumors. However, to enhance its scientific depth and relevance, several key improvements are recommended. While the authors reference viruses such as CMV, JC virus, and Epstein-Barr virus in gliomagenesis, the specific relationship between human papillomavirus (HPV) and glioblastoma multiforme (GBM) is not adequately developed. The introduction would benefit from a more focused discussion of prior studies that have investigated HPV presence in brain tumors, particularly glioblastomas, and from articulating the rationale for exploring this association. The molecular mechanisms by which HPV exerts its oncogenic effects are notably absent. A concise explanation of how HPV oncoproteins E6 and E7 disrupt tumor suppressor pathways—specifically through inactivation of p53 and pRB—would provide essential context. This would help justify the relevance of detecting HPV DNA in GBM tissues and clarify the biological plausibility of its involvement in CNS tumorigenesis. The introduction should more clearly define the knowledge gap this study aims to address. Emphasizing the limited evidence on HPV's role in brain tumors and the need for further investigation would strengthen the framing of the research question and increase the impact of the study. To strengthen the validity and interpretability of the findings, we recommend that the authors include both positive and negative controls in their experimental design and results section. The absence of clearly defined control benchmarks limits the ability to assess the specificity, sensitivity, and reliability of the CISH detection method used for HPV 16/18. Incorporating well-characterized control samples would help clarify the criteria applied for signal classification, intensity grading, and physical state determination, thereby enhancing the rigor of the analysis and supporting the reproducibility of the study. To enhance the robustness of their findings, the authors are encouraged to include an assessment of HPV L1 capsid protein as an indirect means of corroborating the viral presence detected by CISH. The detection of L1 would help confirm active or latent HPV infection and provide additional support for the specificity of the hybridization signals. Furthermore, evaluating p53 protein in the same tissue sections would be highly valuable, as it could offer functional evidence of E6 oncoprotein activity. Given that E6 is known to mediate p53 degradation, observing altered or reduced p53 in HPV-positive GBM tissues would strengthen the argument for a mechanistic link between HPV infection and gliomagenesis. The viral gene expression program of human papillomavirus (HPV) is tightly regulated by the stratification and differentiation of epithelial tissues, particularly in the cervical epithelium where its transcriptional activity is well-characterized. Given this context, the manuscript presents a significant gap by not providing evidence of viral protein expression in glioblastoma tissues, which is essential to support any proposed association between HPV infection and GBM pathogenesis. To strengthen the argument, the authors should either adjust the wording to reflect the limitations of their findings or include immunohistochemical or molecular data demonstrating the presence of viral proteins such as E6 or E7. Furthermore, the assumption that HPV proteins are produced in neuronal or glial cells requires careful justification, as the virus’s expression is known to be highly dependent on epithelial-specific regulatory mechanisms. A discussion addressing how HPV gene expression could occur in non-epithelial tissues like the brain is necessary to clarify the biological plausibility of the proposed link. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Partly References 1. Palomino-Vizcaino G, Bañuelos-Villegas E, Alvarez-Salas L: The Natural History of Cervical Cancer and the Case for MicroRNAs: Is Human Papillomavirus Infection the Whole Story?. International Journal of Molecular Sciences . 2024; 25 (23). Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise molecular diagnostics, aptamer engineering, and nanobiotechnology for viral detection and inhibition, particularly HPV. We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. reply Respond to this report Responses (0) Azuara-Alvarez LE and Vizcaino GP. Peer Review Report For: 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. [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :869 ( https://doi.org/10.5256/f1000research.183561.r421158) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-869/v1#referee-response-421158 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Piranlioglu R et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Oct 2025 | for Version 1 Raziye Piranlioglu , Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA Emerenziana Runco , Brigham and Women’s Hospital, Harvard Medical School, Massachusetts, Boston, USA 0 Views copyright © 2025 Piranlioglu R et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Comments to Authors Zeiny et al. discusses the potential involvement of high-risk human papillomavirus (HPV) types 16 and 18 in glioblastoma multiforme (GBM) in Iraqi patients using chromogenic in situ hybridization (CISH). The authors examined 45 GBM tissue samples and 30 non-neoplastic brain controls and detected HPV 16/18 in 31.1% of GBM and 3.5% of controls. They conclude that infection with HPV may play a role in GBM pathogenesis. We are in favor of indexing; however, this is contingent upon the authors addressing the revisions and concerns outlined below. Major Comments 1. Introduction gives a general overview of glioma and HPV biology but should be improved, setting the focus on more prevalence of HPV infection in Iragi population. Also, the authors should revise their classification of gliomas to align with the most recent updates, as their current description reflects the pre-2016 WHO classification, which was based solely on histogenesis (i.e., the tumor's cellular origin). While this historical approach isn't incorrect, it is now considered outdated in contemporary scientific literature. Additionally, the authors should replace the outdated 2010 prevalence statistics for glioblastoma (GBM) with more current data, as updated figures are now available. Please refer to the sources below: https://seer.cancer.gov/statfacts/html/brain.html https://pubmed.ncbi.nlm.nih.gov/36006639/ Berger et al., (Reference 1) 2. The CISH method is clearly outlined; however, its reproducibility relies heavily on the inclusion of both positive and negative controls. The criteria for interpreting positive signals should be clarified, as distinguishing true positives from false positives is currently challenging. To improve clarity, the authors should present staining results from control groups. A side-by-side comparison showing low, moderate, high, and negative signals from these controls would be valuable for distinguishing signal intensities more effectively. Moreover, the study would be enhanced by a brief description of sample handling (fixation, storage time) and DNA quality control, as paraffin-embedded material can affect viral DNA detection. 3. Determining if the HPV infection is ongoing or latent will enhance impact of study. Since CISH detects viral DNA but cannot distinguish between active and latent infections, the use of qPCR—or RT-PCR—would be helpful to assess viral activity and strengthen the interpretation of results. 4. Does the presence of HPV influence patient survival outcomes or treatment response? Clarifying whether HPV status has any prognostic or predictive value would enhance the clinical relevance of the findings. 5. One important variable not included in this study is the HPV vaccine status of the patients. Since HPV vaccination (for genotypes 16/18) significantly reduces infection rates, information regarding whether GBM and control subjects were vaccinated could be crucial. If these data are available, the authors must analyze whether vaccination is linked to the prevalence of HPV detection. If unavailable, this limitation must be explicitly declared and accounted for as a confounder. Future studies would be enhanced by stratifying patients by vaccination status to ascertain whether detected HPV DNA is attributable to actual infection as opposed to residual viral material not actually involved in disease pathogenesis. 6. The paper appropriately cites several studies but, in most instances, it confuses association with causation. The language must be more precise to avoid the suggestion of a causal link between HPV and GBM. 7. The authors must expand the limitations section: small sample size, retrospective study, paraffin material used, and no molecular confirmation (PCR or RNA-ISH). 8. Have the authors investigated whether there are any genetic differences in the GBM tissues, such as mutations or amplifications of specific genes (e.g., P53 ) that could influence susceptibility to HPV infection? Exploring such molecular alterations may provide important context for understanding the interaction between HPV and tumor biology. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Partly References 1. Berger T, Wen P, Lang-Orsini M, Chukwueke U: World Health Organization 2021 Classification of Central Nervous System Tumors and Implications for Therapy for Adult-Type Gliomas. JAMA Oncology . 2022; 8 (10). Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Cancer Immunotherapy, Brain Tumor Immunology, Oncolytic Virotherapies in Glioblastoma. We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. reply Respond to this report Responses (0) Piranlioglu R and Runco E. Peer Review Report For: 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. [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :869 ( https://doi.org/10.5256/f1000research.183561.r418824) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-869/v1#referee-response-418824 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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