Serum-based studies show novel biomarkers for circulating HPV-DNA in high-risk HPV women as a biomarker for early detection of cervical cancer.

doi:10.21203/rs.3.rs-5361454/v1

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Research Article

Serum-based studies show novel biomarkers for circulating HPV-DNA in high-risk HPV women as a biomarker for early detection of cervical cancer.

https://doi.org/10.21203/rs.3.rs-5361454/v1

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Background:

Cervical cancer caused by HPV is the fourth most common cancer in women worldwide. Detecting circulating cell-free HPV-DNA can provide valuable information about treatment response as well as serve as a diagnostic and prognostic tool for residual disease and relapse. However, only a few biomarkers are available for early diagnosis, and the current ones require tissue excision or invasive procedures, limiting their use in routine diagnosis. Analysis of nucleic acids has become popular for cancer diagnosis, and circulating HPV-DNA is a newly reported biomarker for identifying individuals with cancer. In a recent study, HPV-DNA was successfully detected in 85% of treatment-naive patients, and changes in HPV-DNA levels corresponding to treatment response and relapse were observed. HPV-DNA shows promise as a biomarker for therapy monitoring in both primary and recurrent cervical cancer, and it could become a sensitive, non-invasive, and easily accessible tool for diagnosis and follow-up.

Oncology

HPV-Human papillomavirus

ccf-DNA-Circulating cell-free DNA

Non-Invasive

Circulating normal DNA (cell-free DNA (cfDNA)) refers to DNA fragments that circulate freely in the bloodstream. The origin of fragments can originate from various tissues in the body, including healthy cells.¹ Changes in cfDNA levels can indicate disease progression, treatment response, or recurrence. cfDNA analysis is used for non-invasive prenatal testing (NIPT) to screen for chromosomal abnormalities in prenatal care. The release of circulating cell-free DNA into the blood is triggered by inflammation or infection in the body.² This type of DNA is found in various body fluids, including serum, plasma, blood, saliva, bile, lymph, breast milk, cerebrospinal fluid, and amniotic fluid.³ It is a biomarker for cancer diagnostics, prognostics, and therapeutics and is easily accessible in peripheral blood. Additionally, it is currently being employed in molecular-based research. In a healthy individual, ccf-DNA is released and circulated in minute amounts⁴. However, the necrosis of hematopoietic cells results in the circulation of ccf-DNA. Blood-based molecular diagnostics have become increasingly useful in diagnosing various tumor types, from early diagnosis to treatment monitoring and detecting recurrent disease⁵. Although many cell-free DNA cancer diagnostics have been hindered by less-than-ideal sensitivity, significant progress continues⁶. Virally driven tumors release viral DNA fragments into the bloodstream, making them more easily detected⁷. The potential relevance of using ccf-DNA analysis in clinical oncology, particularly in cancer screening, early diagnosis, therapeutic evaluation, monitoring disease progression, and determining disease prognosis, is gaining attention⁸. In recent years, various technologies have emerged based on the analysis of ccf-DNA for non-invasive prenatal testing, monitoring organ transplantation, and detecting immune diseases and cancers⁹. Apoptosis is a genetically controlled process of self-destruction in cells. It is essential for cellular growth and maintaining homeostasis and plays a role in the pathophysiology of diseases. It is controlled by two pathways and activated by abnormal proliferation and DNA damage¹⁰.

HPV life cycle:

HPV is a small, non-enveloped virus with a diameter of approximately 55 nm. Human papillomavirus is a group of viruses. There are more than 200 HPV types, i.e., 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,59 as carcinogens. HPV16 is 58%, and HPV 18 is 15% prevalent. HPV genome into 5 genera. HPVs are DNA double-stranded spherical Early Region: Contains ORFs (open reading frames) encoding non-structural proteins designated from E1 to E8. Late Genes (L1 and L2): Encode structural viral capsid proteins for virion formation, transmission, and spread. Small viruses with a diameter of about 55 nm and a genome of 8kb. E1 and E2: Involved in viral replication. E4: Induces cytoskeleton rearrangements. E6 and E7 Cause cell-cycle deregulation and are associated with cancer progression. HPV infects only fully differentiated keratinocytes, and the virus amplifies its copy number to around 50–100 copies per cell after infecting wound basal cells. During differentiation, viral gene expression and DNA replication are significantly upregulated, amplifying the viral copy number to thousands of copies per cell. The E6 and E7 genes of HPVs can override cell cycle checkpoints, allowing viral DNA replication and leading to cancer development in rare cases. HPV infects through the skin &mucous membranes, causing vertical transmission from mothers to their newborns during childbirth. Sexual transmission is through direct genital-to-genital contact, oral and anal sex, and repeated use of condoms.

Human papillomavirus (HPV) is a virus that can cause cancer by integrating into the host DNA and disrupting normal cell cycle progression through two genes, E6 and E7. The E6 gene inactivates a protein called p53, which is responsible for preventing the formation of tumors¹¹. The E7 gene allows for the degradation of another protein called pRB, which normally suppresses tumor growth¹². This disruption of the normal cell cycle can cause DNA damage and promote cancer development. Additionally, the E7 gene can induce methylation of the SMG-1 gene promoter, leading to its dysfunction. SMG-1 is a tumor suppressor gene that helps prevent cancerous cell growth¹³.

Existing methods for detecting cervical cancer:

Pap Smear (Pap Test):

The Pap smear is a common screening test used to detect cervical cancer and its precancerous stages. During this test, a healthcare provider collects a sample of cervical cells by gently brushing and scraping the cervix. These cells are then examined under a microscope. An abnormal Pap smear result may indicate the presence of abnormal cells, often caused by HPV infection (Human Papillomavirus), which is a major risk factor for cervical cancer¹⁴.

HPV DNA Test:

The HPV DNA test checks for the presence of high-risk HPV types that are most likely to lead to cervical cancer. It involves testing cells from the cervix to identify any HPV infections¹⁵. A combined screening technique, by using HPV-DNA testing and cervical cytology, increases the detection rates for cervical adenocarcinoma, a very important benefit taking into consideration that, unlike squamous cell cervical carcinoma, the invasive cervical adenocarcinoma incidence has not reduced substantially after the initiation of cervical screening by using cytology¹⁶.

Colposcopy:

If an abnormal Pap smear or HPV test result is found, a colposcopy may be recommended. A doctor uses a colposcope (a magnifying instrument) to closely examine the cervix during a colposcopy¹⁷. This procedure helps identify any abnormal areas that may require further evaluation. Colposcopy provides a detailed view of the cervix, allowing for targeted biopsies, aiding in the correlation of cytological findings with visual examination, and distinguishing between potentially cancerous and benign lesions. Additionally, colposcopy allows for grading lesions and is useful in monitoring treatment effectiveness and detecting recurrence in patients undergoing cervical lesions¹⁸.

Biopsy Techniques:

Punch Biopsy

A small tissue sample is taken from the cervix using a special tool.

Cone Biopsy (Conization)

A cone-shaped tissue is removed from the cervix for examination.

Endocervical Curettage

Cells from the endocervical canal are scraped and examined.

Applications and broader impacts:

Our method applies to highly fragmented and degraded samples like biological samples, clinical samples, FFPE tissue, cell-free RNA/ DNA, liquid biopsies, archaeological samples, and normal samples¹⁹. The variations and errors observed in NGS data due to differential degradation between samples are negligible in our method. This helps generate consistent and high-quality sequencing data²⁰. We could generate 2–10 times more high-quality data with as little as 500pg of total RNA, which is close to the total RNA obtained by a single cell and hence applies to single-cell sequencing²¹. Due to its high efficiency and sensitivity, our method will enable better diagnostics, biomarker discovery, and research. Many biomarker studies are based on the analysis of tumor tissues obtained from invasive surgical procedures and may not be accessible in some cases²². New approaches with non-invasive procedures are urgently needed. ccf-DNA analysis has attracted increasing attention because of its easy accessibility, non-invasive nature, and potential tumor specificity through quantitative detection or specific sequencing²³.

The research community is growing extensively daily, with abundant research in the basic sciences and clinical communities. However, transferring the extraordinary scientific and technological advances in medical research laboratories into care for patients in communities throughout the country has been a major challenge²⁴. Lack of engagement of community practitioners, lack of necessary infrastructure, and the current misalignment of financial incentives and research participation are some of the reasons²⁵. Current evidence shows that the clinical research is too burdensome and has little benefit for the participating clinician or patient. Recommendations for addressing the challenges have included improving the public and political dialog about science, recruiting, training, retaining additional clinical research scientists, and reconfiguring the scientific workforce²⁶. The growing interest in cancer epigenetics is mainly due to the reversible nature of epigenetic changes, which tend to alter during carcinogenesis. Epigenetic regulation plays a critical role in normal growth and embryonic development by controlling the transcriptional activities of several genes²⁷. Over the last two decades, these modifications have been well recognized to be involved in tumor initiation and progression, which have motivated many investigators to research Nutri-epigenetics²⁸. However some synthetic epigenetic inhibitors have been developed for cancer therapy, but their long-term use is hindered because of their toxicities and the development of tumor resistance in patients²⁹. Cancer is a multifactorial disease developed because of several genetic and epigenetic changes. Epigenetics is a process that involves the alteration of gene expression without changing the DNA sequence³⁰. Epigenetic modulations lead to heritable yet reversible changes in histone or DNA that regulate gene expression independent of DNA sequence. Epigenetic dysregulation is often linked to various human diseases, more specifically, cancer³¹.

The drawback of existing methods for the detection of cervical cancer:

The existing methods for detecting cervical cancer have made significant progress over the years, but they do come with certain limitations. Invasive Procedures, Overdiagnosis³², and Commercial DNA isolation kits have been preferred because of their simplicity of application and the shorter time required to achieve high purity and high yield of DNA³³. However, using these commercial kits for screening is expensive and requires expert technical staff. Additionally, there is always a risk of product discontinuation. Numerous high-throughput, cost-effective, and in-house methods have been developed to isolate ccf-DNA from serum samples for PCR applications. For this reason, it is very important to develop new in-house ccf-DNA isolation methods with high yield and low cost, especially for middle-income countries. Our protocol competes with the commercial isolation kit DNA in terms of yield concentration. High isolation efficiency and minimal and easy workflow, which are the minimum requirements of a kit and not considered in the previous kits, have been achieved in our protocol.

Research Methodology

a) Study design: Cross-sectional study

b) Study place:

I. Centre of Excellence in Molecular Biology and Regenerative Medicine, Dept

of Biochemistry.

ii. Pathology Laboratory, JSS Hospital, Mysuru.

iii. Bharat Cancer Hospital College, Mysuru. (MOU with JSSH and BCH)

c) Study Samples: 5ml Blood sample taken from each patient (3ml Red tube & 2ml EDTA tube), Cervical wash specimens collected using an endo-cervical brush.

d) Sample collection duration: Oct-2022 to Oct-2024.

Methods:

In-house preparation of reagents, Phenol chloroform isoamyl alcohol to isolate ct-DNA from cervical wash specimens collected using Endo-cervical brush.
The Standardization of in-lab extraction procedure of ccf-DNA from a plasma sample.
General methods for DNA extraction.
Currently, for standardisation, 30 samples were collected from patients and processed the sample according to the in-lab procedures protocol; methods of DNA extraction are varied immensely. Most published procedures employ a digestion buffer treatment which contains tris salts.

There are five basic steps in the DNA extraction procedure.

Collection of cells or specimens of interest from which DNA is to be extracted.
Lysing the cell open to expose DNA and other nucleic acids with protein.
Adding a concentrated salt solution to make debris such as proteins, lipids and RNA clump together.
Centrifugation to separate the clumps from the DNA.
DNA precipitation and dissolving in a slightly alkaline buffer or nuclease-free water.

The samples could be treated with RNAse and proteinase solution to eliminate contamination. Additionally, a step to purify the extracted DNA could also be included. Generally, successful DNA isolation requires four essential steps: effective disruption of cells or tissue; denaturation of nucleoprotein complexes; inactivation of nucleases; and maintaining the quality & integrity of the isolated DNA, which will directly affect the results of successful scientific research. The three successfully employed DNA extraction techniques are a salting out method using NaCl, laboratory available of the Phenol Chloroform Isoamyl alcohol (PCI) method. The PCI extraction method is the most commonly used DNA extraction method in the literature. Although newer procedures have been developed in recent years, the PCI method continues to be used routinely; the PCI method is employed in our study as well. Molecular studies are highly dependent on the quality and quantity of the extracted nucleic acid. Hence, it is quintessential to develop an extraction procedure that is suitable for molecular processes. The goal of this study was to conduct a proof-of-principle study as a new protocol for DNA extraction from blood-circulating tumor DNA based on the existing PCI method, which could be further used in molecular analyses.

Nucleic acid purification:

We employed the most common, cost-effective Phenol-Chloroform-Isoamyl alcohol (PCI) to purify the DNA. Using buffer-saturated PCI mixture (24:25:1) and high-speed centrifugation (14,000 rpm for 5 minutes), a biphasic phase was generated. DNA and RNA remain in the upper aqueous phase, whereas proteins, lipids, and polysaccharides are sequestered in inter and the organic phases, respectively. The aqueous phase is collected and subjected to further purification using only a Chloroform-Isoamyl alcohol mixture. This ensures the removal of any phenol residues which will affect the integrity of the DNA. After the high-speed centrifugation, again, the aqueous layer is collected and subjected to precipitation.

DNA precipitation:

The aqueous layer obtained from the second phase separation was subjected to precipitation. Ethanol precipitation is the most used technique for concentrating and de-salting nucleic acid preparations in an aqueous solution. The basis of this technique is that when salt and ethanol are added to the aqueous solution, it forces the precipitation of nucleic acids out of the solution. After the precipitation, the DNA can be separated from the rest of the solution by centrifugation. The role of the salt here is to neutralize the charges on the sugar-phosphate backbone. A commonly used salt is sodium acetate. So, in this protocol, we used 1/10 volume of sodium acetate-ethanol mixture to precipitate the DNA. After adding Na-acetate & ethanol mixture, the tubes were placed in a -20⁰C freezer as the reduced temperature will aid in faster precipitation. The precipitated DNA was recovered by centrifugation at full speed for 10mins.The ethanol in the supernatant was discarded, and the pellet was washed with 70% ethanol. Washing with ethanol makes sure that none of the salt contaminants thrives. Again, the tubes were subjected to ice cold centrifuge to pellet down the DNA. Ethanol is removed without disturbing the pellet and air-dried. Removing ethanol entirely is essential, as any leftovers can inhibit further PCR applications. Once the tubes were free of ethanol, the pellets were resuspended in nuclease-free water. The amount of nuclease-free water added here was directly proportional to the size of the pellet.

Data Analysis and Interpretation:

The PCI protocol yielded cell-free circulating DNA (ccf-DNA) in varied amounts, ranging from 4 ng/µL to 740 ng/µL, as determined using a Nanodrop spectrophotometer. However, quality assessment via conventional agarose gel electrophoresis failed to detect this low quantity of ccf-DNA. Consequently, we adopted a PCR-based target amplification method to assess the quality of the isolated ccf-DNA. Data analysis revealed prominent bands for GAPDH and β-Globin, indicating that PCR-based target amplification is an effective strategy for evaluating the quality of ccf-DNA. This method ensures the reliability of our ccf-DNA samples for downstream applications, enhancing the robustness of our diagnostic and research protocols.

Polymerase Chain Reaction (PCR) for HPV DNA

DNA for the amplification of house-keeping gene, GAPDH. Other primers are used for detection of HPV DNA MY09/11, HPV16 & HPV18. All the DNA samples extracted from the Cervical scraping fluid were subjected to PCR.

The PCR was using a brief master mix was prepared containing 0.5U/µL of Taq polymerase (Himedia), 0.6 µM of dNTPs, 10x buffer with 25mM MgCl2 (working = 1x buffer)-3µL and 0.2µM of each of forward and reverse primers. DNA concentration ranging from 50-5030 ng/reaction was added and the total reaction was made upto 30µL using PCR grade water. Amplification was performed in the automated Thermal cycler (Mastercycler gradient, Eppendorf) at 950C for 5min, followed by 35 cycles of 940C for 1 min, 59.50C for 45 sec, 720C for 1 min and a final extension of 720C for 2 min. DNA from known cell lines(HeLa for HPV18 PC and SiHa as HPV 16 PC) were used as positive control(PC) and to check the adequacy of the program. PCR grade water was used as negative control.

The study "Evaluation of circulating HPV-DNA and Circulating Protein in high-risk HPV E6/E7 seropositive women as a biomarker for early detection of cervical cancer" delves into an area of paramount importance in oncology and gynecology. This research occupies a critical space in the ongoing efforts to improve early detection strategies for cervical cancer, a leading cause of morbidity and mortality among women worldwide. The central focus of this investigation is the assessment of circulating HPV-DNA and proteins associated with the high-risk human papillomavirus (HPV) types, specifically the E6 and E7 oncoproteins, in women identified as seropositive for these high-risk HPV strains. The rationale behind exploring these biomarkers stems from the established role of high-risk HPV types in the pathogenesis of cervical cancer, with a significant proportion of cases attributed to the oncogenic activities of HPV E6 and E7 proteins. These proteins are known to disrupt normal cell functions, leading to cellular transformation and potentially developing malignancy. Therefore, detecting circulating HPV-DNA and E6/E7 proteins in blood samples could offer a non-invasive, easily accessible method for early cancer detection, potentially before clinical symptoms or abnormal Pap smear results manifest. The study likely incorporates a series of methodologies in evaluating these biomarkers, including polymerase chain reaction (PCR) for HPV-DNA detection and various protein assay techniques for E6/E7 protein quantification. The sensitivity, specificity, and overall predictive value of these biomarkers for identifying early-stage cervical cancer or pre-cancerous lesions would be crucial determinants of their clinical utility. This research not only contributes to the expansive literature investigating the early detection of cervical cancer but also highlights the potential of biomarker-based screening approaches as complementary or alternative methods to the current standard screening practices, such as Pap smears and HPV testing. Given the non-invasive nature of circulating biomarker detection, this approach could enhance screening acceptability and accessibility, leading to earlier diagnosis and treatment, thereby improving patient outcomes. However, translating these findings into clinical practice demands rigorous validation in diverse populations, alongside evaluations of cost-effectiveness and implementation challenges. Moreover, the ethical considerations surrounding the psychological impact of testing positive for cancer-associated biomarkers on individuals without clinical disease manifestations cannot be overlooked. In conclusion, this study offers valuable insights into the feasibility of using circulating HPV-DNA and E6/E7 proteins as biomarkers for the early detection of cervical cancer. It represents a significant step forward in the quest for improved screening and diagnosis approaches, which could ultimately pave the way for a reduction in cervical cancer incidence and mortality. Further research is essential to refine these biomarkers' utility and integrate them into the broader context of cervical cancer prevention and control strategies.

ETHICS: JSS MEDICAL COLLEGE ETHICS COMMITTEE APPROVED THE STUDY

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Version 1

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You are reading this latest preprint version

Serum-based studies show novel biomarkers for circulating HPV-DNA in high-risk HPV women as a biomarker for early detection of cervical cancer.

Status:

Version 1

Abstract

Introduction

HPV life cycle:

Existing methods for detecting cervical cancer:

Pap Smear (Pap Test):

HPV DNA Test:

Colposcopy:

Biopsy Techniques:

Applications and broader impacts:

The drawback of existing methods for the detection of cervical cancer:

Methodology

Methods:

Nucleic acid purification:

DNA precipitation:

Results

Data Analysis and Interpretation:

Polymerase Chain Reaction (PCR) for HPV DNA

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1