STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This work was supported in part by funds from the federal government (NIMHD grant number 5R44 MD014911-03). Therefore, the federal government has certain rights in this invention.

FIELD OF THE INVENTION

[0002] This invention relates to diagnostic, screening, and early detection methods for cervical cancer, which can also be used to monitor therapeutic effectiveness and relapse monitoring in cancer linked to Human Papilloma Virus (HPV) infection.

BACKGROUND OF THE INVENTION

[0003] The coronavirus disease 2019 (COVID-19), an infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the first global pandemic of the 21 ^st century. Several genomic epidemiology tools have been developed to track the public and population health impact of SARS-CoV-2 community spread worldwide.

More than 137 million cases and close to 3 million deaths have been reported since the beginning of the pandemic in January 2020 (https://coronavirus.ihu.edul A SARS-CoV-2 Variant of Concern (VOC), known as 20I/501Y.V1, VOC 202012/01, or B.1.1.7, was detected in the United Kingdom in November 2020 and has now spread to multiple countries worldwide. Genomic epidemiology studies reveal B.1.1.7 possesses many non- synonymous substitutions of biological/immunological significance, in particular Spike mutations HVA69-70, N501Y and P681H, as well as ORF8 Q27stop and ORF7a. B.1.1.7 shows increased transmissibility and has rapidly become the dominant VOC in the United States (US) (https://covid.cdc.gov).

[0004] The HVA69-70 mutation is a deletion in the SARS-CoV-2 21765-21770 genome region that removes Spike amino acids 69 and 70. The HVA69-70 causes target failure in the TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) assay, catalog number A47814 (TaqPath). TaqPath is designed to co-amplify sections of three SARS- CoV-2 viral genes: Nucleocapsid (N); Open Reading Frame lab (ORF lab)’, and Spike (S). The Spike HVA69/70 deletion prevents the oligonucleotide probe from binding its target sequence, leading to what has been termed S gene dropout or S gene target failure (SGTF). SGTF is associated with significantly higher viral loads in samples tested by TaqPath. S gene target late amplification (SGTL) has also been observed in a subset of samples having Cycle threshold values for S gene >5 units higher than the maximum Ct value obtained for the other two assay targets: N and ORF lab.

[0005] The US and countries where B.1.1.7 rapidly became the dominant SARS- CoV-2 variant require immediate and decisive action to minimize COVID- 19 morbidity and mortality. However, the US does not have a national genomic epidemiology surveillance network for COVID-19 whole genome sequencing (WGS) program in place. Therefore, only a small fraction of all new cases is being sequenced ad-hoc. SGTF has been shown to correlate with the A69-70 mutation highly. Evidently, SGTF can be used as a proxy to monitor SARS-CoV-2 lineage prevalence and geo-temporal distribution and may be near-direct measure of B.1.1.7. [0006] SARS-CoV-2 invades target cells by binding to angiotensin-converting enzyme (ACE) 2 and modulates the expression of ACE2 in host cells. ACE2, a pivotal component of the renin-angiotensin system, exerts its physiological functions by modulating the levels of angiotensin II (Ang n) and Ang-(l-7). ACE2 is widely expressed in the ovary, uterus, vagina and placenta. Therefore, apart from droplets and contact transmission, the possibility of mother-to-child and sexual transmission also exists. To date, COVID-19 has not been reported to be sexually transmitted.

[0007] Loss of ACE2 promoter DNA methylation linked to ACE2 upregulation has been observed in colon adenocarcinoma, kidney renal papillary cell carcinoma, pancreatic adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, and lung adenocarcinoma. DNA methylation dysregulation may also facilitate viral entry, viremia, and an excessive immune response to SARS-CoV-2.

[0008] Widespread testing of asymptomatic people is critical to identify infected individuals and help inform individual quarantine efforts and overall management guidelines for such highly infectious and long-shedding viruses such as SARS-CoV-2, particularly among asymptomatic residents of Black and Latino communities, who are most susceptible and at high risk for SARS-CoV-2 morbidity and mortality. Although nucleic acid-based tests can reveal the presence of the virus, the host epigenome response can provide needed unique information about host and SARS-CoV-2 dynamics. Epigenome modulation by SARS-CoV-2 infection is bound to impact infectivity, morbidity and mortality trends in infected individuals. Molecular testing is a rapid means to determine if someone has been exposed to SARS-CoV-2, and also play an important stratification for patients that are less likely to have any virus being shed, and thus likely less contagious or not a risk at all.

[0009] However, SARS-CoV-2 testing services are not yet been offered in cervical cancer screening clinics. Consequently, we do not have incidence or prevalence data of SARS-CoV-2 infection among asymptomatic women who are seen in cervical cancer clinics in the US. We argue that the approximately 40 million women who annually receive a PAP test result in the US, should be co-tested for SARS-CoV-2 in order to identify asymptomatic COVID-19 patients. This opportunity will also allow us to study if host DNA methylation markers of cervical dysplasia are modulated by SARS-CoV2 infection in HPV+ women.

[00010] The stability of our genome and correct gene expression is maintained to a great extent thanks to a perfectly preestablished pattern of DNA methylation and histone modifications. In cancer and other chronic diseases this scenario breaks down due a sudden loss of global methylation associated with histone modifications which lead to genomic instability, chromosomal rearrangements, activation of transposable elements and retroviruses, microsatellite instability and aberrant gene expression. In cancer an interesting gene-specific phenomenon following global DNA hypomethylation has been widely studied whereby the regulatory regions (CpG islands) of certain tumor suppressor genes (such as BRCA1, hMLH1, p16 ^INK4a , and VHL) become hypermethylated, inactivating the gene as a consequence, whilst the regulatory regions of proto-oncogenes become hypomethylated thus leading to transcriptional activation of the oncogene. Thus global DNA hypomethylation is usually seen together with gene-specific hyper and hypomethylation in cancer and other chronic diseases. The global methylcytosine content of a large collection of normal tissues and tumors has been studied to begin to understand this mechanism in cancer and other diseases.

[00011] The human epigenome is dynamic, not only throughout the cell cycle and during mitotic divisions, but also in its response to environmental factors, which can be critical in development and during aging. Transient and fixed epigenetic modifications continually modulate the normal human epigenome throughout the life course in response to endogenous and exogenous stimuli. The epigenome serves as an interface between the dynamic environment and the inherited static genome, configured during development to shape the diversity of gene expression programs in the different cell types of the organism by a highly organized process. It is has been shown that exposure to physical, biological and chemical factors, as well as exposure to social behavior, such as maternal care, modifies the epigenome. Therefore, exposures to different environmental agents throughout the life course may lead to interindividual phenotypic diversity, as well as differential susceptibility to disease and behavioral pathologies.

[00012] The responses of the epigenome to environmental exposures throughout the life-course are not just aberrations leading to pathology but a biological mechanism that serves as a medium for the adaptability of the genome to altered environments during life. External exposures, physical, chemical, biological and physical exposures received at different levels of social organization lead to changes in the extracellular environment of developing or mature somatic cells, activating signaling pathways, which link extracellular environmental exposures and epigenetic machineries. [00013] The epigenomic machineries are the biological substrate that serves as a mediator between endogenous and exogenous stimuli at different levels of biological organization and the resultant gene expression, which leads to adaptive or reactive responses to said stimuli. The interaction between the internal or external environment and the epigenome is exposure, tissue and cell specific. Therefore, environmental stimuli lead to changes in gene expression levels by interacting with epigenetic machineries without altering the sequence of DNA bases. This interaction leads to a modulation in biological and/or psychological processes that modulate gene expression, in transient and permanent fashion through-out the life-course: from womb to grave. The interaction between non-genotoxic environmental stressors and environmental health promoters and the epigenome occurs at different pathways and intersections of cellular, organ, systemic and bodily functions; from memory formation and synaptic plasticity to adaptation to changing environments.

[00014] DNA methylation, the most important epigenetic modification known, is a chemical modification of the DNA molecule itself, which is carried out by an enzyme called DNA methyltransferase. DNA methylation can directly switch off gene expression by preventing transcription factors binding to promoters.

DETAILED DESCRIPTION OF THE INVENTION

[00015] The present invention, provides in one embodiment, a method for detecting

SARS-CoV-2 nucleic acids in a subject at risk of cancer linked to Human Papilloma Virus (HPV) infection, comprising the steps of: (a) isolating RNA from a specimen; (b) using reverse transcription process to convert the RNA into cDNA; and (c) quantifying SARS- CoV-2 nucleic acids using Real-Time Polymerase Chain Reaction (RT-PCR), Loop Mediated Amplification (LAMP) or SARS-CoV-2 Whole Genome Sequencing (WGS); and (d) comparing the amount of SARS-CoV-2 nucleic acids in a sample to the presence of HPV DNA or RNA (predetermined level), wherein the HPV has been previously quantified, whereby if the value of SARS-CoV-2 nucleic acids in the sample is high in HPV positive subjects, then the subject has an increased risk of having cancer.

[00016] As used herein, the terms test subject, subject or patient are used interchangeably and refer to a human or another animal species, including primates, rodents (i.e. mice, rats, and hamsters), farm animals, sport animals and pets. In one embodiment, the subject is a human. In certain embodiments, the methods find use in experimental animals, in veterinary application, and/or in the development of animal models for disease.

[00017] In another embodiment, the present invention provides, a method for detecting increased risk of having or developing cancer in a subject, comprising the steps of: (a) isolating a RNA sample from a subject at risk of cancer linked to HPV infection; (b) converting the RNA into cDNA; (c) quantifying the expression of SARS-CoV-2 S, N, ORF and/or E genes using Real-Time Polymerase Chain Reaction (RT-PCR), Loop Mediated Amplification (LAMP) or SARS-CoV-2 Whole Genome Sequencing (WGS); and (d) comparing the levels of SARS-CoV-2 gene expression in a sample to the presence of HPV DNA or RNA (predetermined level), wherein the HPV has been previously quantified, whereby if the expression value of SARS-CoV-2 genes in the sample is high in HPV positive subjects, then the subject has an increased risk of having cancer. [00018] In another embodiment, the present invention provides that a cancerous cell or a pre-cancerous cell in a background of SARS-CoV-2 infection has differential DNA methylation values when compared to a cancerous cell or a pre-cancerous cell that is not in a background of SARS-CoV-2 infection, as determined by the presence of SARS-CoV- 2 nucleic acids.

[00019] In another embodiment, the present invention provides that a DNA methylation signature can derived from healthy cells or a healthy tissue is altered in a background of SARS-CoV-2 infection, as determined by the presence of SARS-CoV-2 nucleic acids.

[00020] In another embodiment, the present invention provides a method for screening for increased risk of having or developing cancer in a subject with SARS-CoV-2 infection, as determined by the presence of SARS-CoV-2 nucleic acids, comprising the steps of: (a) isolating a DNA sample from a subject; (b) measuring a DNA methylation signature in a sample; (c) measuring the presence of SARS-CoV-2 nucleic acids; and (d) comparing the values of DNA methylation across the epigenome in a sample with a background of SARS-CoV-2 infection to a the DNA methylation signature in samples taken from a healthy subject or a pool of subjects without SARS-CoV-2 infection, whereby if the value of DNA methylation in the sample is different in the subject with SARS-CoV-2 infection, than the DNA methylation value in subjects) without SARS- CoV-2 infection, then the subject has an increased risk of having cancer.

[00021] In another embodiment, the present invention provides a method for assessing the risk of having cancer in a subject, comprising the steps of: (a) isolating a DNA sample from a subject; (b) measuring a DNA methylation signature in a sample; (cO measuring the presence of SARS-CoV-2 nucleic acids; and (d) comparing the values of DNA methylation across the epigenome in a sample with a background of SARS-CoV-2 infection to a the DNA methylation signature in samples taken from a healthy subject or a pool of subjects without SARS-CoV-2 infection, whereby if the value of DNA methylation in the sample is different in the subject with SARS-CoV-2 infection, than the DNA methylation value in subjects) without SARS-CoV-2 infection, then the subject has an increased risk of having cancer.

[00022] In another embodiment, the present invention provides a method for screening for increased risk of having or developing cancer in a subject with SARS-CoV-2 and HPV infection, as determined by the presence of SARS-CoV-2 and HPV nucleic acids, comprising the steps of: (a) isolating a DNA sample from a subject; (b) measuring a DNA methylation signature in a sample; (c) measuring the presence of SARS-CoV-2 nucleic acids; (d) measuring the presence of HPV nucleic acids; and (d) comparing the values of DNA methylation across the epigenome in a sample with a background of SARS-CoV-2 and HPV infection to a the DNA methylation signature in samples taken from a healthy subject or a pool of subjects without SARS-CoV-2 and HPV infection, whereby if the value of DNA methylation in the sample is different in the subject with SARS-CoV-2 and HPV infection, than the DNA methylation value in subjects) without SARS-CoV-2 and HPV infection, then the subject has an increased risk of having cancer.

[00023] In another embodiment, the present invention provides a method for assessing the risk of having cancer in a subject, comprising the steps of: (a) isolating a DNA sample from a subject; (b) measuring a DNA methylation signature in a sample; (c) measuring the presence of SARS-CoV-2 nucleic adds; (d) measuring the presence of HPV nucleic acids; and (d) comparing the values of DNA methylation across the epigenome in a sample with a background of SARS-CoV-2 and HPV infection to a the DNA methylation signature in samples taken from a healthy subject or a pool of subjects without SARS-CoV-2 and HPV infection, whereby if the value of DNA methylation in the sample is different in the subject with SARS-CoV-2 and HPV infection, than the DNA methylation value in subjects) without SARS-CoV-2 and HPV infection, then the subject has an increased risk of having cancer.

[00024] The methods of the invention comprise using samples that are biospecimens collected from patients or subject animals; samples that are biospecimens collected from a cell culture, and biospecimens collected from a tissue culture.

[00025] In certain embodiments, the methods described herein comprise detecting cancer or any increased risk of having or developing cancer. The cancer can be any neoplastic disease, including carcinoma and solid tumors. The term "cancer" is also meant to include metastatic disease, metastases, and metastatic lesions, which are groups of cells that have migrated to a site distant relative to the primary tumor. In one embodiment, the cancer is a solid tumor. In another embodiment, the cancer is characterized by comprising a metastatic cancer cell population.

[00026] In another embodiment, the cancer is cervical cancer. In another embodiment, the cancer is oropharyngeal cancer. In another embodiment, the cancer is anal cancer. In another embodiment, the cancer is penile cancer. In another embodiment, the cancer is vaginal cancer. In another embodiment, the cancer is vulvar cancer. [00027] In another embodiment, a standard cell or tissue is a non-cancerous cell or tissue. In another embodiment, a standard cell or tissue is a non-neoplastic cell or tissue. In another embodiment, a standard cell or tissue is a non-cancerous differentiated or non- differentiated cell or tissue. In another embodiment, the standard is derived from non- cancerous differentiated or non-differentiated cells or tissues. In another embodiment, the sample and standard are derived from a common cell or tissue but from different sources wherein the standard is derived from a non-cancerous tissue. In another embodiment, the sample and standard are derived from a common tissue but from different sources wherein the standard is derived from a non-cancerous tissue and the sample is from a subject having cancer or suspected of being afflicted with cancer. In another embodiment, the sample and standard are derived from a common tissue and a common source wherein the standard is derived from a non-cancerous cells and the sample is derived from cells suspected of being cancerous cells.

[00028] In another embodiment, the sample is collected after a surgical treatment. In another embodiment, the sample is collected after a radiation therapy. In another embodiment, the sample is collected after a chemotherapy treatment. In another embodiment, the sample is collected before a surgical treatment. In another embodiment, the sample is collected before a radiation therapy. In another embodiment, the sample is collected before a chemotherapy treatment. In another embodiment, a sample is collected before and after a surgical treatment. In another embodiment, a sample is collected before and after a radiation therapy. In another embodiment, a sample is collected before and after a chemotherapy treatment. [00029] In another embodiment the subject has epigenetic changes as a result of exposure to stressful biopsychosocial causal factors, such as, but not limited to, diseases associated to elevated allostatic load, which is linked to the social environment of poor inner-city neighborhoods, remote poor rural areas or marginalized urban sectors that lack social cohesion and have high rates of criminality, abandoned buildings, drug addiction and poverty.

[00030] In another embodiment, an epigenetic change in a subject indicates that the subject has an increased risk of being afflicted with cancer. In another embodiment, an epigenetic change in a subject indicates that the subject has an increased risk of developing cancer.

[00031] In another embodiment, the present invention is used as a genomic/epigenomic cancer screening and/or detecting tool for early detection of every cancer site/type linked to HPV infection. In another embodiment, the present invention is used as an epigenomic cancer screening and/or detecting tool of cancer recurrence after treatment of a primary tumor, as a biomarker of therapeutic effectiveness; and as a biomarker of lifestyle and contextual effects related to cancer prevention, diagnosis and progression of disease. In another embodiment, the present invention provides means to decrease mortality rates, increase survival rates and decrease overall cancer associated health care expenditures, by improving detection, including early detection, detection of recurrences, measuring therapeutic effectiveness and monitoring modifiable lifestyle and contextual effects related to cancer linked to HPV infection. [00032] In another embodiment, the present invention further comprises high throughput detection/screening technology in a clinical setting.

[00033] In another embodiment, the present invention provides that the methods as described herein are used for staging a tumor, thus impacting clinical practice and population cancer incidence and prevalence rates.

EXPERIMENTAL DETAILS SECTION

MATERIALS AND METHODS

EXAMPLE 1: SARS-CoV-2 nucleic acids in cervical liquid cytology specimens

[00034] A proof-of-principle study was performed to ascertain the presence of SARS-CoV-2 nucleic acids in cervical liquid cytology specimens, which had been yested for HP V .

[00035] Automated RNA extraction was performed using the KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head and the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Cat# A42352) or MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit (Cat# A48383) with a sample input volume of 200 μL.

Briefly, we prepared 4 KingFisher™ Deepwell 96 Plates (Cat# A48305) labeled: “Wash 1” (Wash buffer), ‘Wash 2” (80% Ethanol), “Elution solution” and “Sample plate”. To each well of the “Sample plate", we added 5 μL of Proteinase K, 200 μL of each sample, and 200 μL of nuclease-free water to the negative control well. Binding Bead Mix previously prepared, gently mixed five times, and 275 μL added to each sample and the negative control well. Then, 5 μL of MS2 Phage control was added to each well. The MVP_2Wash_200_Flex program was used on the KingFisher™ Flex Magnetic Particle Processor with 96 Deep- Well Head (Cat# 5400630). After the run was completed, the “Elution Plate” was removed from the instrument and covered with MicroAmp™ Clear Adhesive Film (Cat# 4306311). The samples were eluted in 50 μL of Elution Solution, placed on ice for immediate use in real-time RT-PCR assay. The purified nucleic acid was reverse transcribed into cDNA and amplified using the TaqPath™ RT-PCR COVID-19 Kit To prepare the reaction mix, we combined the following components adequate for the number of samples to be tested, in addition to a positive control and a negative control: 6.25 μL of TaqPath™ 1-Step Multiplex Master Mix (No ROX™) (4X), 1.25 μL of COVID-19 Real-Time PCR Assay Multiplex, 7.50 μL of nuclease-free water for a total reaction mix volume of 15.0 μL. Then, we added either 10 μL of purified sample RNA (from RNA extraction), 10 μL of Purified Negative Control, or 2 μL of Positive Control (25 copies/μL of TaqPath™ COVID-19 Control) up to 25 μL of total volume to each well of the reaction plate. We performed the RT-PCR assay using the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument, and the SDS Software vl .4.1, with the following settings: Assay: Standard Curve (Absolute Quantitation), Run mode: Standard 7500, Passive reference: None, and Sample volume: 25 μL. The data was analyzed, interpreted and exported as .csv files using Applied Biosystems COVID-19 Interpretive Software (version 1.3). R (version 4.0.3) was used for biostatistics analyses.

Results

[00036] The results of this study are shown in Figure 1. The median of the SARS- CoV-2 nucleic acid Cycle Threshold (Ct) value was (min-max) for cases and 3.64 (2.86- 4.13) for controls. The standard deviation for the global genomic DNA methylation index was 0.42 for cases and 0.46 for controls and the interquartile range was 1.14 for cases and 1.27 for controls. (Guerrero-Preston, 2007)