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VolitionRx is a multi-national epigenetics company that applies its Nucleosomics TM platform through its subsidiaries to develop simple, easy to use, cost-effective blood tests to help diagnose a range of cancers and other diseases.

We hope that through earlier diagnosis we can help save and improve the quality of human and animals’ lives throughout the world.

Volition is a multi-national epigenetics company that applies its Nucleosomics™ platform through its subsidiaries to develop simple, easy to use, cost-effective blood tests to help diagnose and monitor a range of life-altering diseases including certain cancers and diseases associated with NETosis such as sepsis and COVID-19.

Our mission is to save lives and improve outcomes for millions of people and animals worldwide.

Early diagnosis and monitoring have the potential to not only prolong the life of patients, but also to improve their quality of life.

Our tests are based on the science of Nucleosomics™, which is the practice of identifying and measuring nucleosomes in the bloodstream or other bodily fluid - an indication that disease is present.

We are primarily focused on human diagnostics and monitoring but also have a subsidiary focused on animal diagnostics and monitoring.

We have five key pillars of focus:

Nu.Q ® , Nu.Q ® NETs, Nu.Q ® Capture, Nu.Q ® Discover and Nu.Q ® Vet, all of which use the same proprietary Nu.Q ® platform to commercialize in different areas.

Our research and development activities are centered in Belgium, with an innovation laboratory in California, and additional offices in Texas, London, and Singapore, where we focus on bringing our diagnostic and disease monitoring products to market.

Volition’s Solution and the Science Behind It

Our assays are based on the science of Nucleosomics TM , which is the practice of identifying and measuring nucleosomes in the bloodstream or other bodily fluid, since changes in these parameters are an indication that disease is present.

Background to Genetics, Epigenetics and Cancer

Human genetics, the sequence of our DNA, is essentially a “recipe book” containing details of how to make each of the thousands of different proteins in the human body;

simply put, there is a different gene (or recipe) for each protein.

However, just because a recipe is in the book, does not mean you have to make it, and nobody makes all the proteins in their DNA.

For example, men have all the genes necessary to make ovarian and uterine proteins but do not do so.

Similarly, muscle cells do not make liver proteins or kidney proteins.

This is because the genes for liver and kidney proteins are inactive or “switched off” in muscle cells.

The mechanisms for the control of which genes are active or inactive in a cell are collectively known as epigenetics.

There are many different types of cancers, but generally, the primary cause of a cancer is a mutation within a cell of the DNA encoding or regulating the expression of one or more specific genes called oncogenes.

While many mutations cause no consequence, some can lead to the uncontrolled expansion of the mutated cells and their dissemination to other parts of the body from the tissue of origin in a process called metastasis.

Another consequence of these mutations is an alteration in the epigenetic regulation of many other genes and this, in turn, can create a unique epigenetic signature in the cancer cells.

Epigenetic control is therefore a critical factor in biology and medicine.

A number of epigenetic cancer drugs have been in routine clinical use for more than a decade and the altered epigenetic signature seen in cancer underpins Volition’s diagnostic approaches.

A major mechanism for epigenetic control is mediated through chromosome and nucleosome structure.

Each chromosome contains a long, single molecule of DNA that is coated by a complex array of proteins, mostly in the form of nucleosomes, giving the stretched-out, unwound DNA/protein core, or chromatin, the appearance of “beads on a string.” Unwound chromatin is accessible for reading (or transcribing) and “unwound” genes may be active.

However, genes whose nucleosomes are coiled or supercoiled are inaccessible and inactive.

Figure 1 – A chromosome

We are dedicated to revolutionizing the diagnosis and monitoring of life-altering diseases by advancing the science of epigenetics.

Imagine a world where diseases like cancer and sepsis can be diagnosed early and monitored easily using routine blood tests.

That’s the world we’re trying to build by developing our innovative family of simple, easy to use, cost-effective Nu.Q ® tests.

Our patented Nucleosomics™ technology uses chromosomal structures called nucleosomes as biomarkers in cancer and other diseases:

as explained below, chromosomes consist of the genetic material (DNA) wrapped in a coat of proteins and other molecules.

All the tests in our portfolio detect various characteristic changes in nucleosomes that occur from the earliest stages of disease, enabling early detection and potentially a better way to monitor disease progression and the patient’s response to treatment.

Unlocking Epigenetics

We believe epigenetics is the most exciting field in disease detection and management today.

Modern genetics - the study of genes and heredity, is underpinned by the linear sequences of molecular “letters” present in the DNA double helix of each living cell, many of which encode the genes.

It has had an enormous impact on the practice of medicine, revolutionizing the way doctors identify people with inherited conditions, diagnose cancer, and, increasingly, design personalized treatment plans.

However, there’s more to chromosomes than just the DNA sequence;

at Volition, we focus on chromosomes’ second epigenetic code, which contains a wealth of additional information about the health and function of the body’s cells.

You can think of the DNA sequence of each cell as the text of an instruction manual, and epigenetics as the formatting.

Some parts of the manual are bolded, highlighted, or underlined, telling the cell to emphasize those sections, while others are struck out, telling the cell to ignore those genes.

The cells of most bodily organs are continuously replaced by new ones.

As they die, many old cells release their nucleosomes into the bloodstream.

Our patented Nucleosomics™ technology isolates these circulating nucleosomes from the blood for quantification and analysis.

Chromosome and nucleosome structure represent a major mechanism for epigenetic control.

Each chromosome contains one long, single molecule of DNA that is coated by a complex array of proteins, mostly in the form of nucleosomes, giving the stretched-out, unwound DNA/protein core, or chromatin, the appearance of “beads on a string.” Unwound chromatin is accessible for reading (or transcribing), and unwound genes may be active.

However, genes with coiled or supercoiled nucleosomes are inaccessible and inactive.

Each nucleosome consists of a disc of eight histone proteins wrapped by a short length of DNA.

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Volition’s Epigenetic Approach

Volition’s approach is to investigate the epigenetic structure of chromatin and nucleosomes rather than investigating only the DNA sequence.

We are continuously developing new technologies including:

· A suite of low cost Nu.Q ® immunoassays that can accurately measure nucleosomes containing numerous epigenetic signals or structure, now being developed on a range of different enzyme-linked immunosorbent assay, or ELISA, platforms.

· Nu.Q ® Capture technology to isolate or enrich nucleosomes containing particular epigenetic signals or structures for a wide range of potential scientific and medical applications.

For example, the enrichment of nucleosomes of tumor origin in blood samples taken from cancer patients.

· The production of synthetic (recombinant) nucleosomes, containing exact defined epigenetic signals and structures, is now in-house.

These nucleosomes are used to ensure maximal accuracy of Nu.Q ® immunoassay tests but also have many other applications including Research Use Only, or RUO, kits and as tools in epigenetic drug development.

Improving Outcomes for Cancer Patients

The prospects for cancer patients vary greatly depending on whether the disease is detected at an early localized stage when effective treatment options are available, or at an advanced stage when the disease may have spread, and treatment is much more difficult.

Unfortunately, most cancers are symptomless at early stage and most patients are not diagnosed until the disease has spread to other organs in the body and the likely outcome is poor.

Simple low-cost immunoassay blood tests to detect cancer at an early stage leading to earlier treatment would greatly improve patient outcomes.

The Limitations of DNA Sequencing in Cancer

The advent of next generation sequencing has revolutionized medical research and led to a host of medical and other innovations.

For example, sequencing the DNA of tumor tissue removed by surgery or biopsy uncovers cancer DNA mutations present in the tumor and is used to direct patient treatment selection, but tissue biopsy cannot be used routinely for cancer detection.

However, small fragments of cancer DNA from dead tumor cells are also found in the blood of cancer patients so it is possible to sequence circulating tumor DNA, or ctDNA, in a blood sample taken from a patient to test for any cancer DNA mutations (e.g., mutated P53, KRAS, EGFR).

Unfortunately, these ctDNA blood tests, often called liquid biopsy tests, have thus far also proved ineffectual for early stage cancer detection.

The main reasons why ctDNA tests alone have not proved useful for early cancer detection include:

· The level of DNA fragments circulating in the blood is very low.

· Only a small proportion of the circulating DNA fragments are of tumor origin and the proportion is especially low in early stage cancer (usually less than 1%).

The remaining “healthy” DNA fragments originate mainly from dead white blood cells.

· A DNA sequence mutation will occur on only one in several million (up to 20 million) of the circulating DNA fragments that do originate from cancer cells.

This means that cancer mutations are found in one in millions of a small percentage of a very low level of circulating DNA fragments, with the result that ctDNA is undetectable in most early-stage cancer patients.

· Many cancer-like mutations have recently been found to be present in the blood of healthy elderly people through a process known as clonal hematopoiesis.

Any DNA released from these cells could lead to false positive readings.

Volition’s Epigenetic Approach to Cancer, COVID-19 and Sepsis

Cancer is in essence a disease of genetic and epigenetic mis-regulation of oncogenes and tumor suppressor genes in the chromosomes of affected cells, leading to uncontrolled cell division and eventually to uncontrolled tumor growth and spread.

Thus, the epigenetic signaling structures of chromosomes and nucleosomes are different in cancer cells and healthy cells of the same tissue.

When a cancer cell dies, its chromosomes are digested into nucleosomes as shown in the figure below.

Most nucleosomes are metabolized, but some are released into the blood stream as circulating nucleosomes.

The DNA attached to these nucleosomes is ctDNA.

However, liquid biopsy companies extract only the DNA and discard the remainder of the nucleosome.

Volition analyzes whole circulating nucleosomes containing particular epigenetic signals and structures using our low cost, but highly accurate Nu.Q ® nucleosome immunoassay tests.

Figure 2 – Digestion of a chromosome into nucleosomes.

The epigenetic structure of nucleosomes of cancer origin are known to differ from that of nucleosomes from healthy cells.

These epigenetic changes occur early and drive the development of cancer, for example by inappropriately activating oncogenes that promote cell division or inactivating tumor suppressor genes that repress cell division.

However, the structural epigenetic changes that occur are not restricted to “1 in 20 million” nucleosomes or even to oncogenes and tumor suppressor genes, but are widely distributed, providing a larger cancer signal and enabling earlier detection of cancer.

We use our Nu.Q ® immunoassay tests to detect a variety of early stage cancers.

Circulating cancer nucleosomes also differ from nucleosomes of healthy origin in other ways.

For example, the DNA fragments in cancer nucleosomes are approximately 20 base pairs (or about 14%) shorter than the DNA fragments in nucleosomes originating in healthy cells.

This structural difference is used as the basis of one of Volition’s Nu.Q ® Capture technologies to separate or enrich cancer nucleosomes by removing nucleosomes of healthy origin.

Volition expects that Nu.Q ® Capture technology will further increase the accuracy of its Nu.Q ® immunoassay tests to detect early-stage cancers and will also be useful to ctDNA companies to decrease the cost and increase the accuracy of liquid biopsy tests.

In terms of background science, white blood cells help protect the body against infection.

White cells engulf invading viruses and bacteria and produce antibodies against them.

In addition, white cells also eject chromatin material out of the cell to form Neutrophil Extracellular Traps, or NETs, which catch and trap invading viruses.

NETs are a complicating factor in a wide variety of diseases including respiratory infections, SARS, and pneumonia as well as metabolic diseases, autoimmune conditions, inflammatory conditions, cancer, thrombosis, stroke and sepsis.

While cancer remains our core disease focus, given the prominence of infectious diseases as a result of the pandemic we are also researching the use of our proprietary technology in infectious diseases with particular regard to NETosis and the activation and release of NETs in disease.

In a respiratory infection, white cells migrate to the lungs to protect them from the virus.

Elevated levels of NETs are a clinical complication of the novel coronavirus SARS-CoV-2, or COVID-19, associated with poor patient outcomes.

The ejected NETs material is made up of nucleosomes that can be detected in minute quantities using Volition’s Nu.Q ® nucleosome assays.

Indeed, we believe that we have the only available analytically validated quantitative nucleosome assay.

We believe the versatility of the Nu.Q ® platform and the range of applications for which these assays can be leveraged may help increase diagnostic power and monitor disease progression and potentially treatment response across a range of infectious diseases that involve the over production of NETs, such as COVID-19, pneumonia, influenza and sepsis.

While NETosis is still a relatively new field for Volition, given positive early results, we have formed a Nu.Q ® NETs team to provide increased focus and drive to the product development program.

Our COVID-19 studies in 2020 showed nucleosome levels correlated positively with strong significance:

· with disease severity in the first wave COVID-19 patients,

· with differentiation of patients with mild disease from those admitted to hospitals, and

· between those whom survived or died.

We are now conducting studies of serial testing in second wave COVID-19 patients to determine how predictive our test is and hope to announce additional studies and data throughout 2021.

We continue to research the use of our Nu.Q ® technology for diagnostic and disease monitoring, and as a companion diagnostic to monitor treatment response in a variety of disease conditions.

Research and Development

Volition is developing Nucleosomics TM technologies in a number of areas including:

· Adaptation and optimization of Nu.Q ® immunoassay tests across multiple clinical platforms worldwide for the rapid quantification of epigenetic changes in blood and other biofluids.

Volition’s Nu.Q ® assays for use in clinical studies operate on a random-access immunoassay autoanalyzer approved by the Food and Drug Administration, or FDA, using a chemiluminescent magnetic particle-based assay format, a format which has enhanced analytical performance.

Volition is pursuing both autoanalyzer and manual kit Nu.Q ® platforms for use in its products, services and clinical studies.

· Nu.Q ® assays (both the established plate format and the particle-based format mentioned above) are used for the development of Nu.Q ® blood tests for the most prevalent cancers focusing initially on lung cancer, colorectal cancer and hematological cancers using our Nucleosomics TM biomarker discovery platform.

Our development platform includes assays to be used for asymptomatic (screening) subjects, high-risk populations, symptomatic patients and to monitor disease progression and/or treatment response.

We are developing blood based Nu.Q ® assays to detect specific biomarkers that can be used individually or in combination to generate a profile which forms the basis of a product for a particular cancer or disease.

· Nu.Q ® assays are used for the development of Nu.Q ® blood tests for NETosis.

Our development platform includes assays that may be used for diagnostic purposes and/or to monitor disease progression and/or treatment response.

· Nu.Q ® Capture technology to isolate or enrich nucleosomes containing particular epigenetic signals or structures for complete analysis by mass spectrometry, DNA sequencing, immunoassays or other methods for a wide range of potential scientific and medical applications.

For example, these applications include the enrichment of nucleosomes of tumor origin in blood samples taken from cancer patients for biomarker discovery, and widespread analysis of circulating chromatin fragments that include epigenetically active chromatin proteins.

The Company has also developed the use of the Nu.Q ® technology in veterinary applications and launched its first product, the Nu.Q ® Vet Cancer Screening Test, in the fourth quarter of 2020.

We are in the process of developing additional veterinary products, including a treatment monitoring test, a disease recurrence test and a point-of-care platform.

Our extensive intellectual property portfolio includes the coverage of veterinary applications.

Product Strategy Summary (Human and Canine)

Potential Use

Frontline Human and Canine Screening Tests

Triage Tests to improve sensitivity and/or specificity of existing tests

Aid to Diagnosis

Disease Monitoring

Treatment Response

Asymptomatic Human and Canine Subjects

High Risk Subjects (For example, FIT positive, LDCT positive)

Symptomatic human and canine Patients

Diagnosed human and canine Patients

Treated human and canine Patients

Potential Disease(s)

Most prevalent cancers (Ca) (Lung, Prostate, Colorectal, Breast, Non-Hodgkins Lymphoma, Canine Lymphoma, Canine hemangiosarcoma)

Lung Ca, Prostate Ca, Colorectal Ca

Blood Ca, Colorectal Ca

Canine Blood Cancers

COVID-19, Sepsis,

Through our Nu.Q ® (short for nucleosome quantification) family of tests in our five key pillars, we aim to offer a new, convenient and cost-effective approach to the detection, diagnosis and monitoring of diverse diseases from a simple blood test.

Highlighting abnormalities

Our technology seeks to detect characteristic epigenetic changes in nucleosomes that occur from the earliest stages of cancer and other diseases.

Epigenetic changes often occur before the diseased cells themselves become abnormal enough to show up in traditional biopsies, and oftentimes before the first symptoms are felt.

We aim to replace unpleasant, invasive, and often expensive screening and diagnostic tests such as colonoscopies and biopsies with Nu.Q ® blood tests, helping to save lives and to reduce overall healthcare costs.

Population screening

Our technology and tests could potentially play a game-changing role in early detection of disease in asymptomatic people via routine, population-wide screening.

We believe that simple, cost-effective, and accurate tests are the “holy grail” of an effective screening program.

Risk stratification and diagnostic aid

In addition to being highly informative in their own right, Nu.Q ® tests have the potential to improve the sensitivity and specificity of other clinical tests.

Our tests could also reduce the number of people needing invasive biopsies and other diagnostic procedures, which can be expensive and harmful.

Disease and treatment monitoring

Nu.Q ® tests may act as an early warning system by monitoring treatment response, disease progression and remission.

Improving Outcomes for Patients

We have five key pillars of focus:

Nu.Q ® , Nu.Q ® NETs, Nu.Q ® Capture, Nu.Q ® Discover and Nu.Q ® Vet, all using the same proprietary Nu.Q ® platform.

Nu.Q ® - Detecting cancer early to save lives.

We are developing a simple, cost-effective blood test for cancer.

Cancer is a devastating disease that touches many peoples’ lives, accounting for approximately 10 million deaths worldwide each year.

Early diagnosis is the best way to improve someone’s chances of surviving cancer;

however, current population-wide screening tests (such as mammograms and colonoscopies) are often invasive and unpleasant.

They can also be expensive, causing many people to miss routine screening.

There are no population screening tests at all for some types of cancer, including aggressive forms of the disease such as ovarian or pancreatic cancers.

Unfortunately, many patients are therefore diagnosed too late, when their cancer has already spread, and treatment is more difficult.

We believe that Nu.Q ® can become a cost-effective routine blood test for multiple types of cancer, allowing doctors to check off an extra box along with other routine blood tests like cholesterol during an annual wellness visit.

Nu.Q ® tests have further potential applications in clinical oncology beyond cancer detection.

Being able to use epigenetic information from tumor cells’ nucleosomes could also help physicians select the best treatment for each patient, monitor their response and the disease progression.

We are currently investigating the potential use of Nu.Q ® tests in a range of cancers and clinical settings including:

Nu.Q ® NETs - Monitoring the immune system to save lives.

The immune system can be both friend and foe;

a potent protective force that sometimes overreacts, damaging the body’s own cells and tissues in the process.

We are working to develop tests that will identify people at high risk of poor outcomes/death caused by an immune system overreaction to COVID-19 and other infections.

The immune system is comprised of many different types of white blood cells with different functions.

The most abundant of these white blood cells are neutrophils, which serve as a first line of defense.

When neutrophils detect bacteria, viruses, injuries, or other threats, these cells produce Neutrophil Extracellular Traps (“NETs”), which are sticky webs made of long strings of nucleosomes that work to inhibit a perceived threat from spreading through the body.

Although NETs are an important part of the body’s response to infection, the presence of too many of them in the blood can tip the immune system’s delicate balance between reaction and overreaction.

Elevated levels of NETs are a complicating factor associated with poor patient outcomes in a range of infectious and non-infectious diseases.

Sepsis—widespread tissue and organ damage triggered by an abnormal immune response to an infection—is an area of particular focus for our research on NETs.

A recent global study estimated that there were approximately 49 million cases and 11 million sepsis-related deaths worldwide in 2017, accounting for approximately 20% of all deaths from the same year.

Severe cases of COVID-19 can cause excessive production of NETs in the lungs, which can lead to severe lung impairment or death.

Because NETs contain nucleosomes, our proprietary Nu.Q ® nucleosome assays have been shown to detect NETs.

Using our Nu.Q ® nucleosome assays could enable the stratification of patients with a high level of NETs, allow physicians to rapidly triage these patients, and monitor their disease progression and response to treatment.

The focus on sepsis due to the COVID-19 pandemic has accelerated our research on NETs.

Our current programs include:

Nu.Q ® Capture - Capturing and concentrating samples for more accurate diagnosis - Locating the needle in a haystack.

Human blood is a mixture of many different cell types floating in a complex soup of proteins and other molecules, including nucleosomes released by cells from all around the body.

Detecting a handful of cancerous or other abnormal cells in a patient’s blood sample has historically been like finding a proverbial needle in a haystack.

Volition’s Nu.Q ® Capture program has several strands of technology which either essentially removes background noise, thereby amplifying the signal or looks to identify the signal in a novel way.

This sample enrichment tool removes healthy nucleosomes, leaving an enriched sample of abnormal nucleosomes behind for further analysis.

These nucleosomes contain tumor-specific DNA “typos,” epigenetic changes, and other biomarkers that when analyzed could potentially be used to diagnose a specific type of cancer or other medical condition, guide treatment selection, and monitor disease and treatment progress.

Other strands of Nu.Q ® Capture technology involve isolating various chromatin fragments including nucleosomes and transcription factors from plasma for analysis by mass spectrometry and next-generation DNA sequencing.

Deploying Nu.Q ® Capture as the first blood sample processing step could potentially:

Enhance the sensitivity of subsequent Nu.Q ® immunoassays for diagnosing and monitoring different types of disease using our proprietary Nucleosomics™ platform.

Aid the development of improved diagnostic DNA sequencing methods.

Serve as a quality control tool to reduce the rate of clinical test failure, saving time that is especially valuable for people whose test results are being used to inform their treatment.

Aid the discovery of new biomarkers.

Allow the complete profiling of nucleosomes.

Another novel method utilizing Nu.Q ® Capture and mass spectrometry was published in 2021 and demonstrated the detection and quantification of histone modifications present in the circulating nucleosomes in the blood of cancer patients.

We believe that our work has highlighted for the first time that histone H2A1R3 citrulline is, in plasma, upregulated in colorectal cancer patients and so could be a biomarker we target for future Nu.Q ® immunoassay development.

Furthermore, the use of Nu.Q ® Capture may open up the possibility of using mass spectrometry not only for biomarker discovery as demonstrated in this publication but also as a high throughput platform for screening and/or diagnostics when used in combination with either sequencing and/or our Nu.Q ® assays.

This technology sheds new light on epigenetic changes that cannot be effectively detected amid the noise left behind when using current testing methods, leading to better clinical tests and potentially improved outcomes in the future.

Volition is engaged in multiple research collaborations with academic laboratories working at the cutting edge of their respective fields, to ensure we take advantage of the latest findings and turn them into new clinical tools as quickly as possible.

Nu.Q ® Discover - A complete solution to profiling nucleosomes.

Nu.Q ® Discover gives clinicians, patients, and researchers access to a range of state-of-the-art assays, built on our proprietary Nucleosomics™ platform, for rapid epigenetic profiling in disease model development, preclinical testing and clinical trials.

Our H3.1 assay is also available for purchase as a Research Use Only kit.

Our assays run in our Silver One facility in Belgium or on site and can be used to answer clinical questions, such as measuring treatment efficacy, or on-target and off-target effects in drug development.

Applications include biomarker discovery in oncology, inflammatory conditions, diabetes and more.

Existing and potential customers include both pharmaceutical companies and academic research institutions.

Nu.Q ® Vet (through Volition Vet)

Cancer is the most common cause of death in dogs over the age of two years in the United States.

Up to 50% of all dogs over the age of 10 will develop cancer in their lifetimes.

With approximately 77 million pet dogs in the United States, there are an estimated six million pet dogs diagnosed with cancer each year.

As with humans, earlier detection can save lives and can also improve the quality of life of the dog and its owner.

Yet, as of today, there are few single assay cancer blood tests on the veterinary market.

Currently, dogs suspected of having cancer are required to undergo a variety of diagnostic tests that may be expensive, time consuming, and painful for the animal.

We hope to change this with the introduction of the Nu.Q ® Vet Cancer Screening Test:

a simple, cost-effective, easy to use enzyme-linked immunosorbent assay (“ELISA”) based screening blood test which may help streamline the diagnostic process for older or “at-risk” dogs.

Data have been published in peer-reviewed journals demonstrating Nu.Q ® Vet’s detection of common canine cancers such as lymphoma and hemangiosarcoma.

More recently, data have been presented at the Veterinary Cancer Society Annual Conference suggesting that Nu.Q ® Vet may also serve as a more sensitive measurement of both minimal residual disease and remission and could be a useful monitoring test for dogs with cancer.

We are currently conducting ongoing research regarding Nu.Q ® Vet as follows:

Broadening the Range of Cancer Detected

We have conducted work in other canine cancers and anticipate a further peer reviewed publication in 2022.

Although thus far the Nu.Q ® Vet Cancer Screening Test has been marketed as a screening test for lymphoma and hemangiosarcoma, this Test may be useful in detecting other forms of cancer as well.

The Nu.Q ® Vet Cancer Screening Test performs best for tumors that are more systemic (higher metastatic rate) or have a high cellular turnover rate.

We are also working to incorporate additional histone modifications into the Nu.Q ® Vet Cancer Screening Test that will help to better differentiate between various cancer types.

Differential Diagnosis

We are currently developing additional assays to add to the Nu.Q ® Vet Cancer Screening Test to better differentiate inflammatory and other conditions from cancer.

Studies are underway at five U.S.

university hospitals to collect data comparing a variety of concomitant conditions including:

Inflammatory conditions

Immune mediated disease

Endocrinopathies

Point of Care Test

We are in the process of developing a point of care test to aid the timely provision of diagnosis and treatment response.

Over the next 12 to 24 months, we are planning to explore and evaluate the potential use and early detection efficacy of our Nu.Q ® Vet platform in cats (related to cancer), horses (related to disease and performance fitness), and cattle (for feedlot disease).

Manufacturing Capabilities and Strategy

Our Silver One site in Belgium offers cutting edge, purpose-built manufacturing and processing facilities.

We currently manufacture our own plates and large-scale manufacturing of our antibodies on beads.

Our expert team is on hand throughout, to offer guidance and support and to fulfil customer needs.

Our objective is to establish long-term mutually beneficial commercial relationships.

Commercialization Strategy

Volition believes that given the global prevalence of cancer and diseases associated with NETosis, and the low-cost, accessible and routine nature of our tests, Nu.Q ® could potentially be used throughout the world.

We believe, given the global prevalence of cancer and diseases associated with NETosis, and the low-cost, accessible and routine nature of our tests, Nu.Q ® could potentially be used throughout the world.

We have developed and are continuing to develop a large portfolio of intellectual property (“IP”), centered around the science of identifying and measuring nucleosomes in the bloodstream.

We call this science Nucleosomics™.

Our technologies have a large range of applications, both in humans and animals, to screen, diagnose, and risk stratify patients, and to monitor treatments, disease progression and potential remissions.

While we initially focused on cancer, we have now broadened the range of indications to include several diseases associated with NETosis, including sepsis, which is estimated to be responsible for one in five deaths worldwide.

Our launch sequence is largely determined by the regulatory hurdles we face;

consequently, we aim to initially launch in Europe and Asia, and subsequently in the United States.

We plan to work with partners to commercialize Nu.Q ® worldwide.

Additionally, we are working on complete nucleosome analysis in our Nu.Q ® Capture technology.

The goal of this project is to investigate ways to specifically target ctDNA.

We aim to remain an IP powerhouse in the Nucleosomics™ space and expect to monetize our IP and technologies through licensing and distribution contracts with companies with established distribution networks on a worldwide or regional basis, in both human and animal care.

The first series of products we expect to launch, following the roll-out of our canine cancer screening test, are:

a canine cancer monitoring test;

a NETosis related screening and monitoring test;

Nu.Q ® Discover;

our biomarkers for research purposes and to support clinical trials;

Cancer tests for humans in Non-Hodgkin's Lymphoma, colorectal cancer and lung.

Our Nucleosomics™ technology is transferable to multiple platforms such as ELISA 96-well plates, bead-based chemiluminescent and we are currently working on transferring our technology to the widely-utilized homogeneous immunoassay or HIA platform and several point of care platforms to enable rapid turnaround of results in clinic/the doctor’s office.

Additionally, we are working on complete nucleosome analysis with our Nu.Q ® Capture technology.

The goal of this project is to investigate ways to specifically target circulating tumor DNA (“ctDNA”).

The ability to enrich ctDNA will allow us to use mass spectrometry to analyze histone and DNA modifications, and moreover to sequence the DNA present around the nucleosomes.

This information might enable cancer diagnosis to identify the tissue of origin of that given cancer.

Commercialization will take multiple forms in various markets and opportunities including, but not limited to:

· Direct sales of the Nu.Q ® Vet Cancer Screening Test via the Gastrointestinal Laboratory at Texas A&M University, or TAMU.

· Sales of veterinary clinical products utilizing Nu.Q ® Vet assays and/or Nu.Q ® Capture reagents through distributor networks.

· Licensing of intellectual property, or IP, for clinical products utilizing Nu.Q ® assays and/or Nu.Q ® Capture reagents.

· Sales of clinical products utilizing Nu.Q ® assays and/or Nu.Q ® Capture reagents through distributor networks.

· Licensing of IP for RUO kit sales of Nu.Q ® assays and/or Nu.Q ® Capture reagents.

· Licensing of IP for laboratory developed patient testing services utilizing Nu.Q ® assays and/or Nu.Q ® Capture reagents.

· Provision of direct research services in the processing of samples using Nu.Q ® RUO assays and/or Nu.Q ® Capture.

If we do not have enough funds to fully implement our business plan, we will be forced to scale back our plan of operations and our business activities, increase our anticipated timeframes to complete each milestone or seek additional funding.

In the event that additional financing is delayed, we will prioritize the maintenance of our research and development personnel and facilities, primarily in Belgium.

Our Market Opportunity

Cancer is one of the leading causes of death worldwide, accounting for around 9.5 million annual deaths globally.

There are over 18 million new cases of cancer diagnosed each year and given the aging population this is expected to grow rapidly to over 29.5 million new cases annually by 2040.

Currently, in the United States there are more than three new cases of cancer diagnosed and one person dies from a cancer-related cause every minute.

Statistically, the chances of surviving cancer are greatly improved by early detection and treatment.

However, there are currently very few blood tests for diagnosis of cancer in common clinical use.

Volition believes that early, non-invasive, accurate cancer diagnosis remains a significant unmet medical need and a significant commercial opportunity.

For these reasons, cancer diagnostics is an active field of research and development both academically and commercially.

The global in vitro diagnostic medical device, or IVD, market was $64.5 billion in 2017 and is forecasted to reach $93.6 billion by 2025, registering a compound annual growth rate, or CAGR, of 4.8% from 2018 to 2025.

The forecasted growth is due primarily to the increasing health care demands of an aging population.

The veterinary market for early stage cancer diagnostics is also large and growing, with approximately 77 million pet dogs in the United States alone.

Cancer in dogs is widespread.

It is the leading cause of death for dogs over the age of 10 years and there are over 6 million new dog cancer diagnoses each year.

As cancer screening is not as commonplace in animal health as it is in human health, we believe blood tests like our Nu.Q ® Vet Cancer Screening Test could transform how veterinarians manage cancer in companion animals.

We believe that our low-cost and easy-to-use ELISA based screening blood test for the early diagnosis of cancer will help streamline the screening process, and improve the treatment and quality of life for up to a third of dogs with malignancies, including common malignancies such as lymphoma and hemangiosarcoma.

These two malignancies comprise approximately 2 million cases out of a total of 6 million annual dog cancer diagnoses in the United States.

The United States is currently the largest veterinary market in the world and requires fewer and smaller clinical studies than the FDA process for human diagnostics.

This generally allows for a much faster route to revenue for veterinary products as compared to human products.

We anticipate that because of their ease of use and cost efficiency, our tests have the potential to become the first method of choice for cancer diagnostics, allowing detection of a range of cancers at an earlier stage than typically occurs currently, and testing of animals who, for reasons such as time, cost or aversion to current methods, are not currently being tested.

The initial veterinary focus is the United States and canines but in time we plan to launch products for other cancers, other species, in other countries and other indications such as disease monitoring and even a point of care test.

Our Competition

Volition anticipates facing competition primarily from other healthcare, pharmaceutical and diagnostic companies such as Exact Sciences Corporation, Guardant Health, GRAIL Inc., Freenome Holdings Inc, CellMax Life, Archer DX Inc., Foundation Medicine Inc., Oncocyte Corporation, OpKo Health Inc., MDNA Life Sciences Inc., Oncimmune Holdings Plc, Abbott Laboratories Inc., Cepheid Inc., Koninklijke Philips N.V., GE Healthcare, Siemens, Gen-Probe Incorporated, MDxHealth SA, Roche Diagnostics, EpiGenomics AG, and Thrive Earlier Detection Corp, and from companies such as Mars Incorporated, and IDEXX Laboratories, Inc.

focused on the veterinary space.

Volition applies its Nucleosomics™ platform through its subsidiaries to develop simple, easy to use, cost-effective blood tests to help diagnose and monitor a range of life-altering diseases for both humans and animals including certain cancers and diseases associated with NETosis such as sepsis and COVID-19.

Given the wide-ranging nature of our products in development we believe that our market opportunity is large.

Based on our calculations, we believe that Volition’s annual total addressable market is approximately $70 billion.

Key assumptions for this market forecast are:

opportunity is calculated based on canine and feline populations that are eligible for screening and monitoring.

Nu.Q ® Discover:

opportunity is calculated using drug pipeline data (registered clinical trial programs) for relevant epigenetic targets.

opportunity is calculated based on average length of stay and estimated hospital admissions and discharges for sepsis.

Nu.Q ® Cancer:

opportunity is calculated based on eligible population for annual screening, target participation rates and incidence/prevalence of specific cancers and risk stratification use cases.

We have assumed the following prices per test:

$120 for the U.S., €45 for Europe, $25 for the rest of the world.

$50 globally.

To the extent that one or more of our assumptions prove incorrect our calculation could be materially impacted.

We anticipate that because of their ease of use and cost efficiency of our tests they have the potential to become the first method of choice for disease detection and monitoring in both humans and animals.

O ur Competition

We anticipate facing competition primarily from other human focused healthcare, pharmaceutical and diagnostic companies such as Exact Sciences Corporation, Guardant Health, GRAIL Inc., Freenome Holdings Inc, CellMax Life, Archer DX Inc., Foundation Medicine Inc., Oncocyte Corporation, OpKo Health Inc., MDNA Life Sciences Inc., Oncimmune Holdings Plc, Abbott Laboratories Inc., Cepheid Inc., Koninklijke Philips N.V., GE Healthcare, Siemens, Gen-Probe Incorporated, EpiGenomics AG, MDxHealth SA, and Roche Diagnostics, and from companies such as Mars Incorporated, IDEXX Laboratories Inc., PetDx, One Health Company (Fidocure) and Vidium Animal Health focused on the veterinary space.

There may also be other companies developing products competitive with ours of which we are unaware.

We predict that our future products will have a competitive edge compared to those offered by competitors on the basis that our tests are developed to be accurate, cost-effective, attractive from a government reimbursement perspective, easy to use, non-invasive, technologically advanced, and compatible with immunoassay systems, based on strong intellectual property and to be used for mass screenings.

We predict our future products will have a competitive edge compared to those offered by competitors on the basis that our tests are developed to be accurate, cost-effective, attractive from a government reimbursement perspective, easy to use, non-invasive, technologically advanced, and compatible with immunoassay systems, based on strong intellectual property and to be used for mass screenings.

Many of our competitors have substantially greater financial, technical, and other resources and larger, more established marketing, sales and distribution systems than we have.

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The healthcare industry, and thus our business, is subject to extensive federal, state, local and foreign regulation.

Some of the laws pertinent to our business have not been definitively interpreted by regulatory authorities or the courts, and their provisions are open to a variety of subjective interpretations.

Some of the pertinent laws have not been definitively interpreted by the regulatory authorities or the courts, and their provisions are open to a variety of subjective interpretations.

In addition, these laws and their interpretations are subject to change.

Both United States federal and state governmental agencies continue to subject the healthcare industry to intense regulatory scrutiny, including heightened civil and criminal enforcement efforts.

As indicated by work plans and reports issued by these agencies, the federal government will continue to scrutinize, among other things, the marketing, labeling, promotion, manufacturing and export of diagnostic health care products.

Our diagnostic products fall within the IVD medical device category and are subject to FDA clearance or approval in the United States.

The federal government also has increased funding in recent years to fight healthcare fraud, and various agencies, such as the United States Department of Justice, the Office of Inspector General of the Department of Health and Human Services, and state Medicaid fraud control units, are coordinating their enforcement efforts.

In Europe, medical devices are regulated by self-certification through the Conformité Européenne, or CE, mark system.

Under the system, developers and manufacturers must operate a quality system and validate medical devices in a limited clinical trial to demonstrate the manufacturer has met analytical and clinical performance criteria.

We have implemented an International Organization for Standardization standard, or ISO 13485, Medical Devices – Quality management systems – Requirements for regulatory purposes, or ISO 13485.

The standard addresses managerial awareness of regulatory requirements, control systems, inspection and traceability, device design, risk and performance criteria, as well as verification for corrective and preventative measures for device failure.

Medical device companies such as ours are subject to pre-market compliance assessments from notified bodies, a European Union certification organization which the national authority, or the competent authority, of a European Union member state designates to carry out one or more of the conformity assessment procedures.

ISO 13485 certification establishes conformity to specific European Union directives related to medical devices and allows CE marking and sale of the device.

The European Commission’s In Vitro Diagnostic Regulation 2017/746, or the EU IVDR, became effective in May 2017, marking the start of a transition period for manufacturers selling IVD devices into Europe.

The EU IVDR, which replaces IVD Directive 98/79/EC, has a transition period of five years, after which the EU IVDR will apply in full, and no new applications pursuant to the former directive will be accepted.

Manufacturers have the duration of the five-year transition period to update their technical documentation and processes to meet the new, more stringent European Union regulatory requirements.

We believe the most challenging changes under the EU IVDR will be those regarding the classification of products, which will bring almost all IVDs under the direct review and control of notified bodies, and the performance evaluation of IVDs, which will require extensive clinical and analytical performance studies but also demonstration of scientific validity.

Additional requirements will apply to reinforce the safety of the products, such as extended responsibilities of the economic actors of the supply chain, increased post-marketing surveillance activities, unannounced audits from notified bodies, implementation of an improved traceability and transparency of the devices with, in particular, the introduction of the Unique Device Identification, or UDI, system and an expanded European Database on Medical Devices.

Notified bodies can begin auditing to the EU IVDR once they have been designated as a notified body under the EU IVDR by their competent authority.

TÜV SÜD will be one such notified body.

In practice, it will not be possible to CE mark a product according to the EU IVDR beforehand.

We expect our devices will be deemed to be Class C devices under the EU IVDR, and the conformity assessment procedure for such devices will be a combination of quality management system audits and technical documentation assessments.

The assessment time needed for a technical documentation assessment of a Class C device is expected to be between 2 to 6 months.

We have commenced discussions with the TÜV SÜD to ensure compliance with the EU IVDR as soon as possible.

Regulatory Approach

Commercialization of our future products in the clinical IVD market (e.g.

, for patient diagnosis in hospitals, clinics, etc.) requires government approval (CE marking in Europe, FDA approval in the United States, and Chinese Food and Drug Administration, or CFDA, approval in China).

In the United States, Volition anticipates that its tests will have to be cleared through the FDA’s premarket notification, or 510(k) process, or the FDA’s premarket approval, or PMA, process.

for patient diagnosis in hospitals, clinics, etc.) requires government approval (CE marking in Europe, FDA approval in the United States, and Chinese Food and Drug Administration (“CFDA”) approval in China).

Our diagnostic products fall within the IVD medical device category and are subject to FDA clearance or approval in the United States.

We anticipate our tests will have to be cleared through the FDA’s premarket notification (“510(k)”), process, or its premarket approval (“PMA”) process.

The determination of whether a 510(k) or a PMA is necessary will depend in part on the proposed indications for use and the FDA’s assessment of the risk associated with the use of the IVD for a particular indication.

A similar system operates in China through the CFDA.

In the European Union, our tests can be marketed after a declaration and marking that the test conforms to the essential requirements of the relevant European health, safety and environmental protection legislation, or CE marking.

The CE mark is also recognized in certain Asian territories, including India, for the private payer market.

In Europe, IVD medical devices are regulated by the European Directive 98/79/EC (“EU IVDD”), where products not listed in ANNEX II, such as the ones developed by Volition, can be CE marked through a self-certification process.

Under this system, manufacturers must operate a Quality System and build/maintain a technical documentation file demonstrating the conformity of the product with the requirements of the EU IVDD.

This includes the validation of the devices in a limited clinical trial to demonstrate the manufacturer has met analytical and clinical performance criteria.

The manufacturer then issues a declaration of conformity and affixes the CE mark logo to the product.

In May 2017, the new European In Vitro Diagnostic Regulation 2017/746 (“EU IVDR”), became effective, marking the start of a transition period for manufacturers selling IVD devices into Europe.

The date of application of the EU IVDR, which replaces the EU IVDD, is May 26, 2022.

After this date, no new applications pursuant to the former EU IVDD will be accepted.

We believe the most challenging changes under the IVDR will be those regarding the classification of products, which will bring almost all IVDs under the direct review and control of designated assessment organizations (“Notified Bodies”), and the performance evaluation of IVDs, which will require extensive clinical and analytical performance studies in addition to a demonstration of scientific validity.

Additional requirements will be applied to reinforce the safety of the products such as extended responsibilities of the economic actors of the supply chain, increased post marketing surveillance activities, unannounced audits from Notified Bodies, implementation of an improved traceability and transparency of the devices with the introduction of the Unique Device Identification system and an expanded European Database on Medical Devices.

In January 2022, the EU IVDR was amended.

The May 26, 2022, date of application of the EU IVDR remains unchanged.

Tailored transitional periods have, however, been introduced for devices that must undergo a conformity assessment involving Notified Bodies for the first time under the EU IVDR.

The length of the transitional periods depends on the classification of device.

In practice, the conformity assessment procedure for our products will be a combination of Quality Management System (“QMS”) audits and Technical Documentation assessments.

The time needed for a Technical Documentation assessment of a device by our Notified Body (“TÜV SÜD”) is expected to last for nine months at a minimum.

We have already begun discussions with the TÜV SÜD to ensure compliance with the EU IVDR as soon as possible.

To support the conformity to both the EU IVDD and the new IVDR, Belgian Volition has implemented a QMS, conforming to the internationally agreed standard ISO 13485 that sets out the QMS requirements specific to the medical devices industry.

Belgian Volition has maintained its ISO certification since 2015.

We will also be required to comply with numerous other federal, state, and local laws relating to matters such as safe working conditions, industrial safety, and labor laws.

We may incur significant costs to comply with such laws and regulations in the future, and lack of compliance could have material adverse effects on our operations.

We believe we have structured our business operations to comply with applicable legal requirements.

However, it is possible that governmental entities or other third parties could interpret these laws differently and assert otherwise, which could have a material adverse impact on our business.

Intellectual Property

Volition is developing clinical products based on the enrichment and analysis of epigenetically modified circulating nucleosomes using immunoassay, mass spectrometry, DNA sequencing and other methods.

We have used this position to build a growing, broad, strong and exclusive patent portfolio around the ability to profile the epigenetic environment surrounding circulating chromosome fragments from diseased cells including the epigenetic signaling status of nucleosomes, DNA, and other epigenetic chromatin proteins.

We have used this position to build a growing, broad and strong patent portfolio covering the ability to profile the epigenetic environment surrounding circulating chromosome fragments from diseased cells, including the epigenetic signaling status of nucleosomes, DNA, and other epigenetic chromatin proteins.

Our patent portfolio includes 29 patent families and a total 84 patents granted related to our diagnostic tests (including veterinary applications), with 12 patents granted in the United States, 14 patents granted in Europe, and a further 58 patents granted worldwide.

Additionally, we have a total of 90 patent applications currently pending, with 11 patent applications in the United States, 8 in Europe and a further 71 patent applications worldwide.

Additionally, we have a total of 93 patent applications currently pending, worldwide.

We intend to continue our development of the Nucleosomics™ technologies and will continue to apply for patents for future product developments.

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The patents on the technologies underlying our products should provide broad coverage for each product, including protection through at least 2031.

As of December 31, 2020, we had 65 full-time equivalents compared to 50 as of December 31, 2019.

As of December 31, 2021, we had 83 full-time equivalent (“FTE”) compared to 60 as of December 31, 2020.

We continually assess employee turnover, recruitment initiatives, compensation and benefits programs, safety in performing critical laboratory work, diversity and other matters relevant to human capital management, and we review results with our board of directors on a periodic basis.

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Corporate History

The Company was incorporated on September 24, 1998 in the State of Delaware under the name “Standard Capital Corporation.” On September 22, 2011, the Company filed a Certificate for Renewal and Revival of Charter with the Secretary of State of Delaware.

Pursuant to Section 312 of Delaware General Corporation Law, the Company was revived under the new name of “VolitionRX Limited” (which name was subsequently amended to reflect “VolitionRx Limited”).

The Company acquired its wholly owned operating subsidiary, Singapore Volition Pte.

Limited, a Singapore registered company, or Singapore Volition, on October 6, 2011.

Singapore Volition has one subsidiary, Belgian Volition SRL, a Belgium private limited liability company, or Belgian Volition, which it acquired on September 22, 2010.

Belgian Volition has four subsidiaries, Volition Diagnostics UK Limited, which was formed on November 13, 2015, Volition America, Inc., which was formed on February 3, 2017, Volition Veterinary Diagnostics Development LLC, which was formed on June 3, 2019, and Volition Germany GmbH (formerly Octamer GmbH, or “Octamer”), a Munich, Germany-based epigenetic reagent company that it acquired on January 10, 2020.

VolitionRx Limited was originally incorporated on September 24, 1998 in the State of Delaware under the name “Standard Capital Corporation.” VolitionRx acquired its wholly owned operating subsidiary, Singapore Volition Pte.

Limited, a Singapore registered company (“Singapore Volition”) in October 2011.

Volition Global Services SRL, a Belgium private limited liability company (“Volition Global”), was formed in August 2021, which is a wholly owned operating subsidiary of VolitionRx.

Singapore Volition has one subsidiary, Belgian Volition SRL, a Belgium private limited liability company (“Belgian Volition”), which it acquired in September 2010.

Belgian Volition has four subsidiaries, Volition Diagnostics UK Limited, a private limited company formed under the laws of England and Wales (“Volition Diagnostics”), which was formed in November 2015, Volition America, Inc., a Delaware corporation (“Volition America”), which was formed on in February 2017, Volition Veterinary Diagnostics Development LLC, a Texas limited liability company (“Volition Vet”), which was formed in June 2019, and Volition Germany GmbH (formerly Octamer GmbH, or “Octamer” and now “Volition Germany”), a Munich, Germany-based epigenetic reagent company that it acquired in January 2020.

Our principal executive office is located at 13215 Bee Cave Parkway, Suite 125, Galleria Oaks B, Austin, Texas 78738.

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See our consolidated financial statements and accompanying notes to the consolidated financial statements included in this Report.

WHERE YOU CAN GET ADDITIONAL INFORMATION

We file Annual Reports on Form 10-K, Quarterly Reports on Form 10-Q, and Current Reports on Form 8-K pursuant to Section 13(a) or 15(d) of the Exchange Act electronically with the SEC.

You can access these reports and other filings electronically on the SEC’s website, www.sec.gov .

Compared sentence by sentence after normalising whitespace, quotation marks, case and digits, so re-formatting and restated figures do not read as changed language. Wording changes appear as one removal and one addition. The current filing and the prior one are authoritative.

What changed 10-K

Item 1. Business