The Expression of HPV E6/E7 mRNA in Situ Hybridization in HPV Typing-negative Cervical Cancer

doi:10.21203/rs.3.rs-1076524/v1

Background: High-risk human papilloma virus (HR-HPV) persistent infection is the major tumorigenesis factor for cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). However, the incidence of HPV-negative CC is approximately 5%-30% with different HPV detection methods. HR-HPV E6/E7 mRNA in situ hybridization (RISH) can detect HPV-driven tumours. Our study aimed to explore whether HPV typing-negative CC was caused by HPV infection.

Methods: The records of CC patients with HPV typing results who had undergone cervical biopsies, cervical conization or hysterectomies were collected from April 2018 to September 2021 at the Affiliated Hospital of Weifang Medical University. RISH was detected using RNAscope chromogenicin in the tissues of patients with CC. Immunohistochemistry (IHC) was performed to evaluate the expression of p16INK4a and Ki67. CC patients with positive HPV typing in the same period were collected as controls.

Results: A total of 308 women with HPV typing results were enrolled, and 30 (9.74%) cases of HPV typing were negative, including 22/30 (73.3%) cases of squamous cell carcinoma (SCC) and 8/30 (26.7%) cases of adenocarcinoma. In the CC patients who were HPV typing-negative, 28/30 (93.3%) were positive for RISH and p16INK4a block+ staining, which contained 22/22 (100%) SCCs and 6/8 (75%) adenocarcinomas. RISH was positive in 278/278 (100%) HPV typing-positive CCs, which included 232/232 (100%) SCCs and 46/46 (100%) adenocarcinomas. While 273/278 (98.2%) showed p16INK4a block+ staining, the five negative cases were SCC. Positive RISH in HPV typing-negative CC was significantly lower than in the HPV typing-positive group (P=0.002, 95% CI: 0.848-1.027). However, this significant difference only existed in adenocarcinoma, and positive RISH in HPV typing-negative SCC was the same as in the HPV typing-positive group. No significant differences were seen in the expression of p16INK4a and Ki67 (P=0.291, 95% CI: 0.863-1.047 and P=0.174, 95% CI: 0.905-1.033).

Conclusions: HPV typing may cause misdiagnosis in 9.74% of CC patients, and HPV E6/E7 mRNA can detect the majority of CC with HPV-related status even in HPV typing-negative patients. This approach could provide a novel option to accurately detect HR-HPVs in cervical tumours and help to eliminate the percentage of misdiagnosed HPV-related cases.

Cancer Biology

cervical cancer

HPV typing-negative

HPV E6/E7 mRNA in situ hybridazation

HPV detection

accurate detection

A causal link between persistent infection with high-risk human papilloma viruses (HR-HPV) and the development of cervical cancer (CC) is well established and is the cause of 99.7% of CC and more than 75% of cervical adenocarcinoma [1,2]. Therefore, testing for HR-HPV has been proven to be effective in screening for CC [3,4,5]. However, in the literature, the incidence of HPV-negative CC is approximately 5%-30% and may be related to differences in HPV testing methodology resulting in a high number of false-negative cases [6,7,8]. Therefore, accurate detection of HR-HPVs in cervical lesions is crucial. Current HPV detection methods, which include hybrid capture-II (HC-II), HPV DNA in situ hybridization (HPV DISH), polymerase chain reaction (PCR) and HPV RNA in situ hybridization (HPV RISH), have varying levels of sensitivity and specificity. The HC-II test, which was a homogeneous hybridization assay using unmarked single strand RNA probes compatible with the targeted sequences, was practical, but it could not identify the various genotypes. HPV typing assay (PCR) is the most commonly used HPV DNA detection method for persistent HPV infection, which is the key cause of CC. HPV typing tests have been classified according to the expression of HPV L1 capsid proteins, which are one of the main targets of the cellular immune response and are influenced by HPV DNA integration into the human genome. A reduction or loss of capsid antigen production can result in a reduction in the cellular immune response, and previous research revealed that the reduction in L1 capsid antigen was higher in high-grade squamous intraepithelial lesions (HSILs) than in low-grade squamous intraepithelial lesions (LSILs). HPV typing negativity in CC may be associated with HPV L1 capsid protein loss. Moreover, it cannot distinguish HPV transcriptionally active infections from those defined as “passenger” HPV.

The accumulated data indicated that the viral E6 and E7 proteins are the key factors that maintain the malignant phenotype of HPV-positive cancer cells [9]. It is worth pointing out that E6 and E7 are the only viral genes that are always retained and expressed in HPV-positive cancer cells. HPV-positive cancer cells are oncogene addicted in that their growth is dependent on sustained E6/E7 expression. The overexpression of HR-HPV E6/E7 genes is the key causative factor for CINs and CC. Quantitative reverse transcriptase PCR to detect HPV E6/E7 mRNA would seem to be an ideal HPV testing method since it reveals that HPV is not merely present but is transcriptionally active. The Food and Drug Administration (FDA) approved PCR to detect E6/E7 mRNA as the “gold standard” for the detection and typing of HPV. However, this assay can be performed exclusively in fresh-frozen tumour tissue and lacks the capacity to specify the detected genotypes; as a result, its use is limited in clinical practice. RNA in situ hybridization (RISH) for detecting HPV E6/E7 mRNA transcripts represents an advance in HPV testing because of the ability to detect the virus in its active transcriptional status in formalin-fixed paraffin-embedded (FFPE) tumour tissues. Leading research has shown that RISH is a highly specific and sensitive method for detecting HPV in oropharyngeal squamous cell carcinoma [10]. Additionally, our previous research proved the accuracy of RISH in diagnosing cervical lesions [11].

Herein, we describe a cohort of CC patients with negative HPV typing on FFPE material and explore the expression of HPV E6/E7 mRNA in situ hybridization (RISH). RNAscope technology is a new generation of single-molecule RISH analysis technology. A novel probe design strategy and a hybridization-based signal amplification system to simultaneously amplify signals and suppress the background are applied during this procedure. mRNA expression was observed at the single-cell level under a standard bright-field microscope.

Study population and selection

Patients with CC detected by cervical biopsy, conization, or hysterectomies were retrospectively collected from April 2018 to September 2021 at the Affiliated Hospital of Weifang Medical University in China. The exclusion criteria were as follows: 1. Not tested for HPV typing; 2. History of cervical treatment, regardless of physical therapy or excision; 3. Pregnancy status; 4. Chemotherapy and/or radiotherapy; and 5. Resistant to anti-HPV treatment. In the cohort, cervical carcinomas with negative HPV typing were included; CC patients with positive HPV typing in the same period were selected as controls (Figure 1.).

The patients’ clinical data, which included age at diagnosis, smoking history, drinking history, fertility status, menopausal status, surgical method, and pathological diagnosis, were collected and evaluated. Subjects who smoked at least one cigarette a day and continuously for more than six months were considered positive for smoking history. Drinking at least once a month, including social engagements, was considered positive for drinking history.

Depending on the procedure, samples included cervical biopsies, cervical conization and hysterectomies. The tissue blocks were cut into 5-μm sections on positively charged glass slides for the following assays: 1. H&E staining for morphological identification; 2. p16INK4a immunohistochemistry; 3. Ki67 immunohistochemistry; 4. RISH; and 5. Hs-PPIB RISH (housekeeping/positive control).

Morphological evaluation

Two senior pathologists of gynaecology who were blind to the results reviewed the H&E-stained slides independently, and any ambiguity was resolved by coexamination using a multihead microscope. Based on the WHO 2014 criteria, the research subjects contained 254 cases of SCC and 54 cases of adenocarcinoma, which contained usual, mucinous, serous, clear cell, and minimal deviation adenocarcinoma.

Immunohistochemistry (IHC)

IHC was performed on FFPE tissue as per standard protocols, and antibodies against p16INK4a (clone: G175-405, ZSGB-BIO, China) and Ki67 (clone: MIB1, ZSGB-BIO, China) were used. PBS was used in lieu of the primary antibody as a negative control. A positive control slide was used to ensure the validity of the staining procedure. According to the manufacturer’s instructions, the sections were deparaffinized and dehydrated, and antigen retrieval was performed by boiling the slides with ethylene diaminete traacetic acid (EDTA) antigen retrieval solution (pH 9.0) in a pressure cooker for 15 minutes. After blocking endogenous peroxidase with H₂O₂, the sections were incubated with antibodies, followed by incubation with the secondary antibody. The reaction was detected by diaminobenzidine (DAB) and counterstained with H&E. The IHC results were analyzed independently by two pathologists blinded to the samples.

The p16INK4a staining pattern was classified as negative (no staining), patchy (patchy+, focal and uneven staining in the nuclei and cytoplasm) and block-like (block+, diffuse and even staining in the nuclei and cytoplasm in 100% of the tumour cells). p16INK4a block-like was considered positive [12]. For Ki67, the cells with nuclear staining were counted in at least 10 fields per slide, and the average was calculated. In the cancer cell nuclear staining only, continuous staining was considered positive.

HPV E6/E7 mRNA In Situ Hybridization (RISH)

RISH was performed using the RNA scope 2.5 HD Detection Reagent-BROWN and the HR-HPV 18 probe cocktail (Advanced Cell Diagnostics, Hayward, USA) with the HybEZ^TM hybridization system (Advanced Cell Diagnostics, Hayward, USA) according to the manufacturer’s instructions. Target-specific probes were used to detect the E6 and E7 genes of HR-HPV 18 genotypes (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82). Hs-PPIB was used as the positive control, while normal epithelial cells in the FFPE block were used as the internal negative control. A positive control that showed effective positive staining in the batch of slides effective was used to ensure that the slide processing was successful. The assay was performed according to the supplier’s instructions (Advanced Cell Diagnostics, Hayward, USA). Pretreatment: After being deparaffinized and dehydrated, these sections were serially treated with Pretreatment 1 and Pretreatment 2 solutions, followed by incubation with Pretreatment 3 solution overnight. Hybridization: The sections were hybridized in HR-HPV 18 hybridization solution without a cover slip in a HybEZ^TM Oven (Advanced Cell Diagnostics, Hayward, USA). Signal amplification: The hybridized probe was performed through the serial application of Amp 1-6. Visualization: DAB was used to demonstrate the amplified signal. The sections were counterstained with H&E, dehydrated with graded ethanol and xylene, and mounted with Cytoseal.

Images of the RISH slides were taken at 20× magnification using a Motic light microscope (Motic BA600 Mot, Germany). A positive RISH test result was defined as positive if any of the malignant cells showed brown punctuate dot-like nuclear and/or cytoplasmic positivity [13,14].

Following identification and screening by H&E staining, the effective nucleus was taken as the core while eliminating the identified oversized adherent nuclei and undersized cell fragments to identify the areas with DAB staining signals within the cell range and to record the positive patterns. To exclude any possible morphological influences, the RISH slides were evaluated by two pathologists who were blinded to the morphological diagnoses.

Statistical analysis

Statistical analyses were performed using SPSS 25.0 (SPSS, IBM). The rate and percentage of expression were used to describe the general situation of the study subject. Chi-square analysis was used to test for differences in expression rate. A P value of < 0.05 was considered statistically significant.

In the total cohort, 308 women with HPV typing results were enrolled, and most CC patients (300/308, 97.5%) had clinical symptoms of contact bleeding, irregular vaginal bleeding or/and abnormal discharge; conversely, some patients (8/308, 2.5%) had no abnormal symptoms diagnosed by the health examination. The clinical characteristics of these patients are presented in Table 1. The mean age at diagnosis was 47.0 ± 9.7 years old, with a range of 26-73 years old. The median age was 46 years old, while the age of most patients at diagnosis was 47 years old. Furthermore, 56 women (56/308, 18.2%) were postmenopausal, and no women were nulliparous. All the patients had a reproductive history of birth at least once, and women of minimum age were included.

Table 1

The clinical characteristics of the CC patients included in the study.

Parameters	Numbers	Percent
Mean age	47.0 ± 9.7	26-73
Smoking history
YES	0	0%
NO	308	100%
Drinking
YES	0	0%
NO	308	100%
Fertility status
Nulliparous	0	0%
Pluriparous	308	100%
Menopausal status
Pre-menopausal	252	81.8%
Post-menopausal	56	18.2%
Presentation
Contact bleeding	300	97.5%
Symptomless	8	2.5%
HR-HPV infection
YES	278	90.3%
NO	30	9.7%
Pathological diagnosis
SCC	254	82.5%
Adenocarcinoma	54	17.5%

HPV typing was negative in 30 cases (9.74%), which included 22/30 (73.3%) cases of squamous cell carcinoma (SCC) and 8/30 (26.7%) cases of adenocarcinoma. The details are presented in Table 2. In the CC patients who were HPV typing-negative, 28/30 (93.3%) were positive for RISH and p16INK4a block+ staining, which included 22/22 (100%) SCCs and 6/8 (75%) adenocarcinomas. RISH was positive in 278/278 (100%) HPV typing-positive CCs, which included 232/232 (100%) SCCs and 46/46 (100%) adenocarcinomas. While 273/278 (98.2%) showed p16INK4a block+ staining, the five negative cases were SCC. Positive RISH in the HPV typing-negative CC group was significantly lower than that in the HPV typing-positive group (P=0.002, 95% CI: 0.848-1.027). However, this significant difference only existed in adenocarcinoma, and the incidence of positive RISH in HPV typing-negative SCC was the same as that in the HPV typing-positive group. No significant difference was seen in the expression of p16INK4a and Ki67 (P=0.291, 95% CI: 0.863-1.047 and P=0.174, 95% CI: 0.905-1.033).

Table 2

Descriptive statistics of the detection of RISH, p16INK4a and Ki67 in different groups.

	Research group		Control group		P value
	SCC	AC*	SCC	AC	P value
RISH
+	22	6	232	46	0.002
-	0	2	0	0	0.002
p16INK4a
+	22	6	227	46	0.291
-	0	2	5	0	0.291
Ki67
+	22	7	232	46	0.174
-	0	1	0	0	0.174
*AC: adenocarcinoma

A total of 232/278 (83.4%) of the patients had a positive HPV typing of SCC, and the positive staining of RISH is presented in Figure 2. The malignant cells showed brown punctuate dot-like nuclear and/or cytoplasmic positivity. p16INK4a positivity showed diffuse and even staining in the nuclei and cytoplasm in 100% of the tumour cells. The representative positive staining of the RISH of adenocarcinoma patients who were positive for HPV typing presented with p16INK4a and Ki67 strongly positive staining, as shown in Figure 2. Additionally, the expression of RISH and Ki67 was clearly positive, but p16INK4a was negative, and no block-like staining was observed in SCC with positive HPV typing, as shown in Figure 2.

The characteristics of the RISH-positive staining of cervical SCC and adenocarcinoma patients who had negative HPV typing presented with positive p16INK4a and Ki67 staining, as shown in Figure 3. Otherwise, the staining of adenocarcinoma was in conformity, with no positive RISH signals presented. Ki67 was also negative, while p16INK4a expressed block-like staining, as shown in Figure 3.

In addition, it is worth noting that adenocarcinoma with no staining of RISH and p16INK4a, in which p16INK4a staining was not expressed in every gland, had no patchy or block-like staining in the nucleus or cytoplasm of cancer cells, while Ki67-positive staining is presented in Figure 3.

The positive percentages for RISH were 93.3% and 100% in the HPV typing-negative and HPV typing-positive groups, respectively (Table 3). The different markers in the same group had no statistically significant differences in RISH, p16INK4a, p16INK4a/Ki67 and different combinations (all P> 0.05) (Table 3).

Table 3

Statistical descriptions of RISH, p16INK4a and Ki67 (+: positive; -: negative.).

	Group		Percent (research)	Percent (control)	P*
	HPV-typing negative	HPV-typing positive	Percent (research)	Percent (control)	P*
RISH
+	28	278	93.3%	100.0%	1.0
-	2	0
p16INK4a
+	28	273	93.3%	98.2%	1.0
-	2	5
p16INK4a/Ki67
+	27	273	90%	98.2%	1.0
-	3	5
RISH/p16INK4a
+	27	273	90%	98.2%	1.0
-	3	5
RISH/p16INK4a/Ki67
+	27	273	90%	98.2%	1.0
-	3	5
*: P: P value compared with different detection methods in the research group.

In this study, we concluded two major findings: 1. HPV E6/E7 mRNA was expressed not only in CC with positive HPV typing but also in the majority of CC with negative HPV typing. HPV E6/E7 mRNA positive expression might reflect the phase of HPV infection, which ‘contributes to’ the development of HPV-associated malignancy. 2. The negative expression of p16INK4a does not exclude HPV infection, and p16INK4a positivity does not necessarily indicate HPV infection.

It is well established that persistent infection with oncogenic HPV subtypes is necessary for the contribution to CC [7] and results in nearly all cases of CC [1]. A multicentre, open-label, randomized clinical trial concluded that the primary screening of HR-HPV for cervical cancer was effective [15], and HPV typing detection is widely used in CC screening programs at present. However, a previous study in SCC using FDA-approved cervical screening technologies showed that HPV testing can be negative in patients with SCC diagnosed by liquid-based cytology [16]. HPV-negative CC is approximately 5%-30% and may be related to differences in HPV testing methodology [6,7,8]. In our study, HR-HPV-negative cases accounted for 9.74% of HPV typing CC, which was nearly consistent with previous studies [6,7,8]. Another study demonstrated that HPV DNA, using different molecular methods in tissue samples, accounted for 45.9% of SCCs, which were previously diagnosed as HPV-negative SCCs on cytology material. Those authors had included that the higher percentage of HPV-negative results in some series was due to the high number of false-negative cases considering sampling variability, low virus load, rapidly progressive cancers developing between cervical cancer screening intervals and the rarity of truly HR-HPV-negative SCC [17]. Recently, a previous study of the integrated genomic and molecular characterization of CC showed that 95% of CCs are HPV-positive [18]. These studies concluded that a subset of patients exists in which HR-HPV is not detectable by current laboratory methods used in the clinic.

In the early stage of HPV infection, the virus is mostly present in a transient or latent status, and the HPV genome is in a free state in the nucleus [11,19]. HPV E6/E7 genes are regulated by E1/E2 genes, and their expression is inhibited during this stage [11,20,21]. HPV E6/E7 mRNA is often undetectable in normal cervical tissues or LSIL [11,22]. During the persistent infection stage, HR-HPV DNA integrates into the host genome with the disruption and loss of the E2 gene, which leads to HPV E6/E7 mRNA overexpression and L1 capsid protein loss [9,23]. In some studies, an in situ hybridization test has been proven to have higher sensitivity and specificity in the detection of HPV infection, and the HPV E6/E7 mRNA test was considered a potential tool [23,24]. In our study, the typical HPV E6/E7 mRNA RISH image of CC presented with weak-to-strong nuclear and cytoplasmic dot-like signals within malignant cells. Our study also showed that a subset of women may have a false-negative HPV typing result that may be associated with fluctuations in HR-HPV positivity and HPV L1 capsid protein loss. Moreover, HPV E6E7 mRNA RISH was negative in 2 (25%) HPV typing-negative cervical adenocarcinomas, which was in accordance with the previous study that CC cases were not all HPV-infected [23,25], and potential contributing factors included infection with an HPV genotype not currently contained in standard testing platforms, variations in sampling, low viral load or decreased transcriptional activity, excluding E6/E7 genes, host p53 and retinoblastoma protein (Rb protein) mechanisms, and metastatic neoplasms in vivo in early or truly HR-HPV-negative carcinoma [6,9,20,23]. In contrast to a previous study, which showed that cervical SCC cases were not all HPV-infected [26], all SCCs in our study were positive for RISH, which could reveal that the HPV E6/E7 mRNA test had superior detection in HPV-associated SCC.

Histopathology is the gold standard for the diagnosis of cervical carcinoma. However, pathologists may use some markers to make a differential diagnosis in some cases with equivocal pathological features, including p16INK4a and Ki67; however, such markers are not specific. p16INK4a is regarded as a reliable surrogate method to detect HR-HPV with a sensitivity approaching 100% [27], and the use of p16INK4a has been reported in several studies on cytology and histologic samples as a biomarker assisting in the diagnosis of high-grade epithelial cervical lesions [28]. In our study, most CCs with p16INK4a-positive staining were 93.3% and 98.2% in the two groups, respectively, which was concordant with previous studies reporting p16INK4a-positive staining from 80% to 100% in invasive carcinoma [29]. The variation in expression rates may partly depend on the criteria defining positive expression. In this research, nuclear and continuous diffuse cytoplasmic staining of the cells was considered positive, while some studies required nuclear or cytoplasmic staining to be positive [29]. However, negative cases were detected in adenocarcinoma. Many previous studies insisted that the overexpression of p16INK4a, as a multitumour suppressor gene, may be closely correlated with HPV oncoprotein E7 inactivating cell cycle regulation Rb protein [30] and was directly involved in the regulation of the cell cycle, inhibiting the activity of cyclin-dependent protein kinase CDK4/CDK6. In our study, we thought that p16INK4a negative staining might be evoked via pathways other than HPV infection through gene mutation or hypermethylation. Currently, the specific mechanism is not fully understood, and the correlation between p16INK4a and HPV infection history remains unclear. It is also controversial whether the p16INK4a expression level can accurately predict the potential risk of precancerous lesions or CC.

Ki67 positivity is highly dependent on reactive changes and cell proliferation [31]. As a nuclear proliferation antigen, Ki67 is exclusively expressed in the proliferation phase of the cell cycle. Some previous data revealed that the Ki67-positive staining percentage in normal cervical tissues and CIN and cervical cancer tissues gradually increases with the aggravation of the disease [29]. In our study, the levels of Ki67-positive staining were 96.7% and 100% in the two groups, respectively. These results were better concordant with previous studies that found Ki67 in 90% to 100% of invasive carcinomas [29]. Ki67 and p16INK4a/Ki67 are the most widely used biomarkers that significantly improve the accuracy of the pathological diagnosis of cervical lesions [32].The cotest of Ki67-positive and p16INK4a-positive staining usually indicates that the cell cycle is out of control, which occurs in abnormal cell proliferation. However, our study showed that Ki-67 and p16INK4a/Ki67 exhibited no significant improvement over p16INK4a alone in the diagnosis of CC. Hence, the routine addition of Ki-67 to p16INK4a was not recommended, which was consistent with the results of previous studies [11]. Additionally, some cases expressed positive staining of p16INK4a, while HPV E6/E7 mRNA was not expressed, a high correspondence between p16INK4a and the RISH test was shown in the literature; however, even in only a few cases, discordant results were found in our study and some previous studies [13,33-35], which may have resulted from HPV detection methods. In other words, the negative expression of p16INK4a does not exclude HPV infection, and p16INK4a positivity does not necessarily indicate HPV infection [36,37].

The present study has several limitations. As a retrospective study, specimens were taken from samples in the past that were not tested by HC-II, HPV DISH and HPV E6/E7 mRNA (APTIMA) to further confirm the presence or absence of HR-HPV. Another limitation was that our study had no detailed history information on HPV infection and had limited available data from Pap screening tests and colposcope examinations, which reflected both the small percentage of patients who were diagnosed with CC who were RISH-negative overall and the fact that the majority of patients who developed CC had inadequate screening. Previous data also revealed that 60% of women diagnosed with CC had never received Pap screenings or had done so at irregular intervals [38]. These data would certainly assist further in the interpretation of the correlation of HR-HPV-associated CC (false-negative) and IHC findings. A multicentre, large-scale and further prospective study with more appropriate and accurate data should be conducted.

HPV typing tests could cause misdiagnoses in approximately 9.74% of CC patients, and HPV E6/E7 mRNA could detect all cervical SCC and most cervical adenocarcinoma with HPV-related status even when HPV typing is negative. This approach could provide a novel opinion to accurately detect HR-HPVs in cervical tumours and help to eliminate the percentage of misdiagnosed HPV-related cases.

abbreviations	full title
CC	cervical cancer
CIN	cervical intraepithelial neoplasia
DAB	diaminobenzidine
EDTA	ethylene diamine tetraacetic acid
FDA	The Food and Drug Administration
FFPE	formalin-fixed paraffin-embedded
HC-II	Hybrid Capture-II
HPV DISH	HPV DNA in situ hybridization
HPV RISH	HPV RNA in situ hybridization
HR-HPV	high-risk human papilloma virus
HSIL	high-grade squamous intraepithelial lesions
IHC	immunohistochemistry
LSIL	low-grade squamous intraepithelial lesions
PCR	polymerase chain reaction
RISH	HPV E6/E7 mRNA in situ hybridization
SCC	squamous cell carcinoma

Acknowledgements

This work is supported by a grant from the National Natural Science Fund of China (No. 81572559) and the Scientific Research Foundation of Weifang Medical University (2017BSQD43).

Authors’ contributions

YT X and YZ L: conception and design of the study, assembly, analysis and interpretation of the data, and manuscript writing. YH S, H C, JJ C, CC C, BG Z and YZ Z: provision of study materials, analysis and interpretation of the data. The author(s) have read and approved the final manuscript.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The study protocol was approved by the ethics committee at the Affiliated Hospital of Weifang Medical University, China.

Consent for publication

All patients gave their consent for their information to be published in Diagnostic Pathology.

Competing interests

The authors have no conflicts of interest to declare.

Author details

^*Correspondence: [email protected]

¹School of Clinical Medicine, Weifang Medical University, No. 7166, Baotong Road, Weifang, Shandong 261053, China

²Department of Obstetrics and Gynaecology, Affiliated Hospital of Weifang Medical University, No. 2428, Yuhe Road, Weifang, Shandong 261042, China

³Department of Pathology, Affiliated Hospital of Weifang Medical University, No. 2428, Yuhe Road, Weifang, Shandong 261042, China

⁴Department of Obstetrics and Gynaecology, Maternal and Child Health Care Hospital of Linzi District, Dashun Road, Zibo, Shandong 255400, China

⁵Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China.

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69(1):7-34. doi:10.3322/caac.21551
Stolnicu S, Hoang L, Hanko-Bauer O, et al. Cervical adenosquamous carcinoma: detailed analysis of morphology, immunohistochemical profile, and clinical outcomes in 59 cases. Mod Pathol. 2019;32(2):269-279. doi:10.1038/s41379-018-0123-6
Bulkmans NW, Berkhof J, Rozendaal L, et al. Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasia grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial. Lancet. 2007;370(9601):1764-1772. doi:10.1016/S0140-6736(07)61450-0
Ogilvie GS, van Niekerk D, Krajden M, et al. Effect of Screening With Primary Cervical HPV Testing vs Cytology Testing on High-grade Cervical Intraepithelial Neoplasia at 48 Months: The HPV FOCAL Randomized Clinical Trial [published correction appears in JAMA. 2018 Dec 4;320(21):2273]. JAMA. 2018;320(1):43-52. doi:10.1001/jama.2018.7464
Dijkstra MG, van Zummeren M, Rozendaal L, et al. Safety of extending screening intervals beyond five years in cervical screening programmes with testing for high risk human papillomavirus: 14 year follow-up of population based randomised cohort in the Netherlands [published correction appears in BMJ. 2016 Oct 27;355:i5782]. BMJ. 2016;355:i4924. Published 2016 Oct 4. doi:10.1136/bmj.i4924
Alexander C, White M, Maleki Z, Rodriguez EF. HPV-ISH-Negative Invasive Cervical Squamous Cell Carcinoma: Histologic and Pap Test Results. Acta Cytol. 2019;63(5):417-423. doi:10.1159/000500595
Tao X, Griffith CC, Zhou X, et al. History of high-risk HPV and Pap test results in a large cohort of patients with invasive cervical carcinoma: experience from the largest women's hospital in China. Cancer Cytopathol. 2015;123(7):421-427. doi:10.1002/cncy.21545
Samimi SA, Mody RR, Goodman S, et al. Do Infection Patterns of Human Papillomavirus Affect the Cytologic Detection of High-Grade Cervical Lesions on Papanicolaou Tests?. Arch Pathol Lab Med. 2018;142(3):347-352. doi:10.5858/arpa.2016-0478-OA
Hoppe-Seyler K, Bossler F, Braun JA, Herrmann AL, Hoppe-Seyler F. The HPV E6/E7 Oncogenes: Key Factors for Viral Carcinogenesis and Therapeutic Targets. Trends Microbiol. 2018;26(2):158-168. doi:10.1016/j.tim.2017.07.007
Randén-Brady R, Carpén T, Jouhi L, et al. In situ hybridization for high-risk HPV E6/E7 mRNA is a superior method for detecting transcriptionally active HPV in oropharyngeal cancer. Hum Pathol. 2019;90:97-105. doi:10.1016/j.humpath.2019.05.006
Hui C, Bai H, Liu J, et al. Accuracy of HPV E6/E7 mRNA examination using in situ hybridization in diagnosing cervical intraepithelial lesions. Diagn Pathol. 2021;16(1):13. Published 2021 Feb 19. doi:10.1186/s13000-021-01072-9
Darragh TM, Colgan TJ, Cox JT, et al. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology [published correction appears in Arch Pathol Lab Med. 2013 Jun;137(6):738]. Arch Pathol Lab Med. 2012;136(10):1266-1297. doi:10.5858/arpa.LGT200570
Mirghani H, Casiraghi O, Amen F, et al. Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice. Mod Pathol. 2015;28(12):1518-1527. doi:10.1038/modpathol.2015.113
Zito Marino F, Ronchi A, Stilo M, et al. Multiplex HPV RNA in situ hybridization/p16 immunohistochemistry: a novel approach to detect papillomavirus in HPV-related cancers. A novel multiplex ISH/IHC assay to detect HPV. Infect Agent Cancer. 2020;15:46. Published 2020 Jul 14. doi:10.1186/s13027-020-00310-x
Zhang J, Zhao Y, Dai Y, et al. Effectiveness of High-risk Human Papillomavirus Testing for Cervical Cancer Screening in China: A Multicenter, Open-label, Randomized Clinical Trial. JAMA Oncol. 2021;7(2):263–270. doi:10.1001/jamaoncol.2020.6575
Austin RM, Onisko A, Zhao C. Enhanced Detection of Cervical Cancer and Precancer Through Use of Imaged Liquid-Based Cytology in Routine Cytology and HPV Cotesting. Am J Clin Pathol. 2018;150(5):385-392. doi:10.1093/ajcp/aqy114
Tao X, Zheng B, Yin F, et al. Polymerase Chain Reaction Human Papillomavirus (HPV) Detection and HPV Genotyping in Invasive Cervical Cancers With Prior Negative HC2 Test Results. Am J Clin Pathol. 2017;147(5):477-483. doi:10.1093/ajcp/aqx027
Cancer Genome Atlas Research Network; Albert Einstein College of Medicine; Analytical Biological Services; Integrated genomic and molecular characterization of cervical cancer. Nature. 2017;543(7645):378-384. doi:10.1038/nature21386
Woodman CB, Collins SI, Young LS. The natural history of cervical HPV infection: unresolved issues. Nat Rev Cancer. 2007;7(1):11-22. doi:10.1038/nrc2050
Münger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 1989;8(13):4099-4105.
Yeo-Teh NSL, Ito Y, Jha S. High-Risk Human Papillomaviral Oncogenes E6 and E7 Target Key Cellular Pathways to Achieve Oncogenesis. Int J Mol Sci. 2018;19(6):1706. Published 2018 Jun 8. doi:10.3390/ijms19061706
Graham SV. The human papillomavirus replication cycle, and its links to cancer progression: a comprehensive review. Clin Sci (Lond). 2017;131(17):2201-2221. Published 2017 Aug 10. doi:10.1042/CS20160786
Chen T, Li J, Wang S, Ning Y, Zhou X, Wang Y. High-risk HPV E6/E7 mRNA in situ hybridization in endocervical glandular neoplasia: performance compared with p16INK4a and Ki67 immunochemistry. Am J Transl Res. 2019;11(10):6498-6506. Published 2019 Oct 15.
Derbie A, Mekonnen D, Woldeamanuel Y, Van Ostade X, Abebe T. HPV E6/E7 mRNA test for the detection of high grade cervical intraepithelial neoplasia (CIN2+): a systematic review. Infect Agent Cancer. 2020;15:9. Published 2020 Feb 7. doi:10.1186/s13027-020-0278-x
Alexander C, White M, Maleki Z, Rodriguez EF. HPV-ISH-Negative Invasive Cervical Squamous Cell Carcinoma: Histologic and Pap Test Results. Acta Cytol. 2019;63(5):417-423. doi:10.1159/000500595
Lewis JS Jr. Human Papillomavirus Testing in Head and Neck Squamous Cell Carcinoma in 2020: Where Are We Now and Where Are We Going?. Head Neck Pathol. 2020;14(2):321-329. doi:10.1007/s12105-019-01117-y
Madrigal E, Bishop JA, Faquin WC. Head and Neck Cytopathology: Human Papillomavirus-Positive Carcinomas, Including Diagnostic Updates, Testing Modalities, and Recommendations. Surg Pathol Clin. 2018;11(3):501-514. doi:10.1016/j.path.2018.04.002
Hodgson A, Park KJ, Djordjevic B, et al. International Endocervical Adenocarcinoma Criteria and Classification: Validation and Interobserver Reproducibility. Am J Surg Pathol. 2019;43(1):75-83. doi:10.1097/PAS.0000000000001095
Kanthiya K, Khunnarong J, Tangjitgamol S, Puripat N, Tanvanich S. Expression of the p16 and Ki67 in Cervical Squamous Intraepithelial Lesions and Cancer. Asian Pac J Cancer Prev. 2016;17(7):3201-3206.
Martin C, Oleary JJ. Histology of cervical intraepithelial neoplasia and the role of biomarkers. Best Pract Res Clin Obstet Gynaecol. 2011;25:605–15.
Goel MM, Mehrotra A. Immunohistochemical expression of MIB-1 and PCNA in precancerous and cancerous lesions of uterine cervix. Indian J Cancer. 2013;50(3):200-205. doi:10.4103/0019-509X.118729
Kanthiya K, Khunnarong J, Tangjitgamol S, Puripat N, Tanvanich S. Expression of the p16 and Ki67 in cervical squamous intraepithelial lesions and cancer. Asian Pac J Cancer Prev. 2016;17:3201–6.
Lewis JS Jr, Ukpo OC, Ma XJ, et al. Transcriptionally-active high-risk human papillomavirus is rare in oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas--a tissue microarray study utilizing E6/E7 mRNA in situ hybridization. Histopathology. 2012;60(6):982-991. doi:10.1111/j.1365-2559.2011.04169.x
Bishop JA, Ma XJ, Wang H, et al. Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. Am J Surg Pathol. 2012;36(12):1874-1882. doi:10.1097/PAS.0b013e318265fb2b
Ukpo OC, Flanagan JJ, Ma XJ, Luo Y, Thorstad WL, Lewis JS Jr. High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma. Am J Surg Pathol. 2011;35(9):1343-1350. doi:10.1097/PAS.0b013e318220e59d
Castle PE, Adcock R, Cuzick J, Wentzensen N, Torrezmartinez NE, Torres SM, et al. Relationships of p16 Immunohistochemistry and other biomarkers with diagnoses of cervical abnormalities: implications for LAST terminology. Arch Pathol Lab Med. 2020;144(6):725–34.
Zafereo M, Xu L, Dahlstrom KR, Viamonte CA, Elnaggar AK, Wei Q, et al. Squamous cell carcinoma of the oral cavity often overexpresses p16 but is rarely driven by human papillomavirus. Oral Oncol. 2016;56:47–53.
Wheeler CM, Hunt WC, Joste NE, Key CR, Quint WG, Castle PE. Human papillomavirus genotype distributions: implications for vaccination and cancer screening in the United States. J Natl Cancer Inst. 2009;101(7):475-487. doi:10.1093/jnci/djn510

No competing interests reported.

The Expression of HPV E6/E7 mRNA in Situ Hybridization in HPV Typing-negative Cervical Cancer

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Study population and selection

Morphological evaluation

Immunohistochemistry (IHC)

HPV E6/E7 mRNA In Situ Hybridization (RISH)

Statistical analysis

Results

Discussion

Conclusion

Abbreviations

Declarations