The analysis of HPV integration sites based on nanopore sequencing and the profiling changes along the course of photodynamic therapy

Four patients with cervical intraepithelial neoplasia complicated with high-risk HPV infection who were admitted to the Department of Gynecology, Beijing You'an Hospital Affiliated to Capital Medical University from January 2021 to December 2021 were selected as the research objects. Two cases’ histopathology of cervical biopsy were CIN I while the other two were CIN II. The cases aged 30–52 years (Table 1). All the cases completed two stages of treatment [3 times of photodynamic therapy (3 PDT) and 6 times of photodynamic therapy (6 PDT)]. All patients were aware of this study and signed informed consent. This study was approved by the Ethics Committee of Beijing You'an Hospital jing you ji lun zi [2019]003.

Based on the analysis of HPV abundance, profiling changes of HPV integration sites and the gene set related to cervical cancer progression after enrichment, we developed a nanopore sequencing based assay as companion diagnostic tools to conduct individualized treatment. As HPV abundances increased, HPV integration sites were found increasing correspondingly, and more genes related to cervical cancer were also observed. The advantage of this assay over traditional qPCR tests, was not only better sensitivity (we can identify enriched viral reads when viral load below the LOD of qPCR) to avoid misdiagnosis, but also quantitatively detection of viral abundance and integration sites at once, moreover, with enriched reads over viral genome, we could more accurately identify viral genotype, sub-genotypes or even drug-resistant variations.

The HPV genome is divided into three regions: (i) the long control region (LCR) which contains binding sites of viral regulatory proteins (E1 and E2) and cellular transcription factors, (ii) the early region (E) that encodes for the E1, E2, E4, E5, E6 and E7 genes and (iii) the late region (L) that encodes for the L1 and L2 genes [6, 20, 21]. Over the last few decades, the strong tumourigenic capacity of HPV16 DNA triggered the extensive sequence analysis of the viral genome, several nucleotide changes have been associated with immune escape, viral persistence and cervical cancer development [22, 23]. HPV16 is a strong carcinogenic factor, because it is detected in more than 50% of cervical cancer cases. The tumourigenic hallmark of HPV16 DNA lies mainly on the function of E6 and E7 oncoproteins and numerous analyses have proved that mutations particularly within E6 increase the oncogenicity of viral DNA [24‐26]. The T350G (L83V) was a nucleotide variation of HPV16 DNA on the E6 gene, implicated in the development of severe dysplasia in various geographic populations. In vitro experiments demonstrated that the coexistence of nucleotide changes G145T (Q14H), C335T (H78Y) along with T350G (L83V) considerably augment the tumourigenic capacity of the viral oncoprotein [27, 28]. To date, no nucleotide variations have been involved in the development of cervical disease concerning E4 gene. Conversely, the nucleotide change A4042G/T (I65V) in the E5 oncogene seems to be mainly identified in specimens diagnosed with advanced cervical dysplasia [29‐31]. The LCR is regarded as the most polymorphic region of viral DNA, with numerous novel nucleotide variations to be continually reported in diverse research studies.

HPV infects the host cells and usually exists in a free state, HPV latent in the basal cell layer, where its DNA moves into the nucleus of infected cells in an independent exogenous chromosomal state. Subsequently, HPV integrates into the host chromosome, leading to genomic instability and tumorigenesis of the host. Upon integration the circular episomal HPV DNA is converted into a linear structure and inserted into the host chromosome. The disruption of viral genome can take place within the early region of viral genome (E1 and E2 genes) [32‐34], or within the late region (L1 and L2 genes) [35, 36]. The most frequent site of disruption within the E2 ORF is located in the portion that encompasses the hinge region of the E2 protein [34, 35, 37]. In contrast, disruptions within E1 gene have been found to be more frequently located either at the 5′ end [32, 38], or at the 3′ end of the E1 gene [36].

HPV is often present in cervical lesion cells with a mixed infection state, for instance, 2 different genotypes of HPV were found in patient B19, with HPV58 in the integrated state and HPV52 in a free state. Integration of HPV into the host chromosome is a key factor in cervical carcinogenesis. Genes related to cervical cancer progression was found by enrichment analysis before treatment, including CTNNB1, CCR7, AKT1, SLIT2, CDK6, etc. Human SAIL4 (sal-like 4), the new discovered proto oncogenes encode transcription factors that acted at the level of stem cells, SAIL4 protein can recognize and bind to CTNNB1 promoter region and trans‐activating CTNNB1 for accelerated expression β- Catenin, which promoted cell proliferation and the formation of tumor cervical cancer cells [39]. Invasion and metastasis of tumor cells through the bloodstream and lymphatic vessels were key steps in the progression of cervical cancer, CCR7 was implicated in mediating lymphocyte trafficking and spreading to lymph nodes, expression of CCR7, CXCR4, VEGF-C and VEGF-D may have a synergistic effect on the malignant development of cervical cancer and lymph node metastasis [40]. Akt1 and p-Akt1 were key elements of PI3K/Akt signaling pathways that regulate cellular processes, including proliferation, differentiation, migration and survival, the HK2 (the member of hexokinases, Associated with malignant tumor growth and distant metastasis) and AKT1 (p-AKT1) may regulated in the same networks in cervical cancer cells and synergistically promoted malignant growth and distant metastasis during the development of cervical cancer [41]. It had been shown that SLIT2 expression levels were negatively correlated with the incidence of cervical cancer in patients treated with postoperative chemotherapy (CT) or radiotherapy (RT), indicating that SLIT2 may be a potential predictive biomarker [42]. Cyclin-dependent kinase6 (CDK6) was a crucial regulatory cancer-related gene within the cell cycle and tumorigenesis, the changes in the level of CDK6 expression may impact on the progression of cervical precancerous lesions in the female [43]. HPV integration frequency increases with severity of cervical precancer, and viral integrations are present in the majority of cervical cancers. In this study, patients B02 and B16, both had a pathological grade of CIN 1, and had a lower pathological grade than patients B15 and B19 with CIN 2, patients B02 and B16 had lower viral abundance and significantly fewer integration sites than the latter.

The principle of photodynamic therapy is to selectively kill actively proliferating cells. The more severe of cervical lesion, the more actively proliferating of its cells, with a greater number of HPV insertion sites and a higher HPV abundance. After photodynamic therapy, the actively proliferating cervical cells are killed and the degree of cervical lesions is significantly reduced. The integrated HPV viruses were cleared, so the HPV abundance and the number of integration sites decreased significantly in patients B02 and B19. However, in patients B15 and B16, we found that although their cervical pathological grade decreased after photodynamic therapy, their HPV abundance and the number of integration sites did not decrease accordingly, considering that the persistent HPV infection lead to new HPV integration. Especially for patient B16, the HPV59 abundance and the number of integration sites increased significantly after 3 PDT, the possible reason for this being the ineffectiveness of PDT and predicting the persistent infection of HPV and a possible exacerbation of the cervical lesions. Subsequent follow-up results also confirmed the presence of persistent infection of HPV56 and HPV59, while the cervical lesions developed from cervicitis to LSIL. Its possible explanation was the production of new HPV integration. Nanopore technology can predict the direction of disease progression earlier than TCT and pathology. Regrettably, patient B16 refused any further HPV integration site testing and pathology testing. We can still identify some genes related to cervical cancer progression from the samples of the two patients after treatment, including ATF3, GPX3, IL10, IL6, RAD51AP1, MGMT, WWOX. Activated transcription factor 3 (ATF3) was a stress-induced transcription repressor, it had been shown that ATF3 overexpression enhanced paclitaxel apoptosis in cervical cancer HeLa cells in part by stimulating TAp73, thus, it can act as a tumor suppress factor [44]. Glutathione peroxidase 3 (GPX3) was the member of the glutathione peroxidase family of selenoproteins, GPX3 promoter methylation was one of the main causes of downregulated GPX gene expression in cervical cancer and acted as a predictive biomarker for lymph node metastasis and prognosis in cervical cancer [45]. In cervical cancer tissues, the expression levels of IL10 and Ki-67 increased accordingly when the HPV infection rate increased, so the detection of IL10, KI67 expression and HPV infection rate can provide reference for the diagnosis of cervical cancer [46]. Thrombocythemia, high PLR (platelet to lymphocyte ratio) and IL-6 overexpression correlated with cervical cancer progression and metastasis, they may play an important role as molecular markers of survival and may be considered as potential predictors of cervical cancer prognosis [47]. RAD51AP1 was the main BRD4 target gene participating in radiosensitivity, and high expression of BRD4 was found to be associated with poorer prognosis and radioresistance. JQ1 (a BRD4 inhibitor) sensitized cervical cancer to radiation therapy by suppressing RAD51AP1 transcription [48]. Studies had shown that 5-Aza-dC (DNA methyltransferase inhibitor) and X-ray radiation can inhibit expression of p16 (cyclin-dependent kinase inhibitor 2A, CDKN2A) and MGMT in cervical cancer cells, and down regulating the expression of p16 and MGMT can promote the anti-proliferation and treatment of cervical cancer cells, this discovery may provide a new treatment and a new method for cervical cancer [49]. It had been shown that WWOX is under-expressed in cervical cancer tissues and cell lines, and the expression of WWOX significantly decreases or disappears with the development of cervical cancer, proved that the reduced expression of WWOX is strongly associated with the development of cervical cancer, regulating the expression of WWOX may be an effective and novel method to treat cervical cancer [50]. Therefore, these two patients are still at high risk of relapse, even the clinical remission were observed. The prognosis of cervical lesions cannot be determined by pathological diagnosis alone, but also by integrating information on HPV abundance and integration profiling changes, especially those HPV integrated genes/networks related to carcinogenesis.

In this study, we developed a nanopore-sequencing based assay that provides information on HPV infection, genotype and integration all at once for patients with cervical lesions along the course of photodynamic therapy. The technique advantages of viral sequence enrichment combined with nanopore long-read sequencing contributed to more accurate prognosis determination as well as the dynamic monitoring of treatment accompaniment for a better evaluation of photodynamic therapy. That makes it a promising companion diagnostic tools and favorable supplement to traditional pathological examinations.