What do the test results tell me?

The results
1) confirm cancer presence
2) give measurable or quantifiable information about disease presence.

The IvyGene Score is calculated as a composite average of cell-free DNA that is methylated at specific sites as a fraction of the total cell-free DNA present. An IvyGene Score of 19 and below is considered normal. An IvyGene Score of 20 or greater is considered elevated.

An elevated IvyGene Score means that the amount of cell-free DNA in the blood that is methylated at specific sites is consistent with the presence of cancer.

The IvyGene Score itself does not provide an outright indication cancer stage as various factors influence the amount of DNA shed by solid tumors (tissue of origin, tumor location, overall tumor burden, etc.). Instead, the IvyGene Score flects the relative amount of cell-free DNA that originated from tumor tissue. The amount of cell-free DNA shed into circulation by malignant cells is predicted to correlate with tumor burden.

Some physicians have advised that an increase or decrease in 2 or more successive IvyGene tests may be an indicator of treatment effectiveness as it can demonstrate a decline in the methylated cell-free DNA at specific sites.

Cancers progress at varying rates and therefore, the frequency of testing is subject to the orderingprovider’s independent clinical judgement.

The IvyGene test does not provide tissue of origin.

How should results be interpreted?

Results should be interpreted by a medical provider and used in conjunction with the patient’s complete medical history.

0-19 is considered a normal result and 20+ is considered an elevated result. These cut-offs were established by comparing samples drawn from healthy subjects, subjects with benign disease and samples drawn from subjects diagnosed clinically confirmed breast, colon, liver, and lung cancers.

The presence of other cancers may result in an elevated IvyGene score.

As with any test, the IvyGene test is not 100% accurate and there is a possibility of false positives and false negatives. The current sensitivity of the IvyGene test is 84% and the specificity is 90%. The sensitivity of the IvyGene test for additional cancer types will be released as we continue to validate new cancer types.

What is the turnaround time for the test results?

Results are returned to the ordering physician via fax or email within 5 business days of the test kit receipt by our laboratory, excluding holidays.

False Positives or False Negatives. What might cause a false positive or false negative?

Certain types of chemotherapy drugs that target DNA methyltransferases (the enzymes responsible for DNA methylation) , may interfere with the results of the IvyGeneTM test. These chemotherapy drugs include: Azacytidine and Decitabine.

Introduction

DNA methylation is an epigenetic regulator of gene expression. In mammals, the DNA base cytosine can be methylated at the C5 position of the cytosine ring to form 5- methylcytosine (5mC). The dynamic methylation of cytosine to 5mC often occurs at sites in the genome where cytosine is immediately adjacent to guanosine, which are called CpG sites (5′-CG-3′). These CpG sites are frequent found in clusters in the promoter regions of genes. Hypermethylation of the CpG sites within a gene promoter is associated with gene silencing.

IvyGene® Test Performance Characteristics

LAM’s CLIA registered and CAP accredited laboratory ran a validation data set performed on samples drawn from 197 subjects with either no history of cancer or a diagnosis of breast, colon, lung and liver cancer. Cancer patients were diagnosed according to current clinical practices. The IvyGene scores of these subjects are presented in Figure 2.

By using a pre-established clinical cutoff of 20 (IvyGene Score ≥ 20 is positive), the IvyGene test was determined to possess a calculated sensitivity of 84% (95%CI: 75- 93), and a specificity of 90% (95%CI: 85-95).

Additional test performance characteristics are summarized in Table 1.

Table.1 Summary of IvyGene Test Performance Characteristics

*Positive and negative predictive values are dependent upon prevalence. The estimated, combined incidence of breast, colon, liver can lung cancers in the general US population is 168 per 100,000

• 0-19 is considered a “Normal Result,” which is not consistent with the presence of cancer.
• This has been validated on breast, colon, liver, and lung cancer samples, in addition to a cohort of healthy individuals.

The specificity of the IvyGene test is 90% Rember: Specificity is the measure of correctly identified Healthy patients

The sensitivity of the IvyGene test is 84%. Remember: Sensitivity is the measure of correctly identified patients with disease

• 20+ is considered an “Elevated Result,” which is consistent with the presence of cancer.
• This has been validated on breast, colon, liver, and lung cancer samples, in addition to a cohort of healthy individuals.
• Other cancer types may cause an elevated result, but until validations are completed for those other cancer types, we would not know the IvyGene Test’s accuracy for confirming those cancer types.

Collection Kit

Instructions for Use

Read all instructions prior to collection

For Investigational Use Only

LAM-CK-001

Indication for Use

The IvyGene® Collection Kit is intended for blood collection, anticoagulation, stabilization, transportation and short-term storage of venous blood plasma samples for preparation of human cell-free DNA for use in molecular diagnostic test for detection of specific CpG methylation patterns.

Storage and Handling Requirements

• Store the lvyGene® Collection Kit at room temperature (1 5°CY 30°C, 60°F ~ 85°F).
• Use the IvyGene® Collection Kit before the “Use By” date printed on the Collection Tubes and Collection Box
• If your lvyGene® Collection Kit is requiring that you to freeze the Refrigerant Pack, please put the Refrigerant Pack in a -20°C freezer (-4°F) for at least 12 hrs. prior to shipping.

DO NOT FREEZE the lvyGene® Collection Tubes i

Collecting the Sample: All International Samples are Pre-pay for Cost and Shipping

1. Check the provided tubes for the expiration date and ensure they are viable.
2. Using the provided [PaxGene] tubes only, draw a sufficient amount of blood to completely fill at least 4 tubes of blood.
3. Invert each tube slowly 5 times.
4. Include the Date of Birth and Unique Identified [included on the requisition form] on ALL 4 tubes.
5. Store the samples at room temperature only. No freezing or refrigeration required.

*Samples have a shelf life of 7 days, stored at room temperature. Do not wait more than 24 hours to ship the sample. Should a sample be received more than 7 days post blood draw, it will be rejected by Quality Control. *

Preparing the Sample

1. Place all 4 sample collection tubes in the provided Biohazard plastic bag, with the absorbent sheet. Remove the white liner of the Biohazard bag to reveal adhesive and seal the plastic bag.
2. Wrap the entire Biohazard bag and samples inside the provided bubble wrap. Then place the biohazard bag and contents inside the white Tyvek envelope and seal.
3. Place the Tyvek envelope inside provided Styrofoam container, between the Room Temperature Packs [do not freeze or refrigerate these packs]. Place the lid on the Styrofoam container with the test requisition form and payment information. Close securely with tape.

*Should you accidentally refrigerate or freeze the packs, they may be placed in warm water until packs become gel-like once more. *

Shipping the Sample

1. Visit Fedex.com to print and pay for shipping.
2. Ship Samples to:
LAM Indiana
1201 Cumberland Avenue
West Lafayette, IN 47906
3. Choose an option that will ship the test kit with sample within 4 business days.
4. Attach the shipping label to the original box with the IvyGene logo.
5. Print and track the shipment information to ensure the shipment is delivered correctly.

*Refunds for test cost and shipping will not be issued for tests shipped incorrectly or that arrive and do not pass QC due to shipping conditions. *

*Should a sample not pass QC at the laboratory for internal reasons, a refund will be issuefor the test cost only. *

Every day thousands of people in the USA are beginning to ght cancer. In 2016, more than 4,600 individuals in the U.S. were diagnosed with cancer daily. The rst step to ghting cancer is identifying its presence early, which can be a di cult and frustrating process.

The lvyGene test is a blood test that uses advanced DNA sequencing methods to detect the DNAmethylation pattern of circulating tumor DNA (ctDNA) in blood samples in order to both confirm the presence of cancer and give measurable information about cancer. The lvyGene test is a non-invasive, accurate tool to aid physicians in confirming cancer presence at the time of testing.

According to studies performed by the National Cancer Institute and the American Cancer Society, individuals who identi ed 25 di erent types of cancer earlier had a greater chance of survival. In combination with diagnostic tests, the IvyGene test is another step towards winning the battle with cancer.

How does the IvyGene Test Work?

The IvyGene® Test measures the methylation status of cell-free DNA extracted from blood samples at target gene sites. These target sites have been demonstrated to be hypermethylated when certain cancers are present. Test results are Reported as a quantitative IvyGene® Score, which indicates the methylation status of the target sites. The IvyGene® Score is calculated as a composite average of cell-free DNA that is methylated at the target sites as a fraction of the total cell-free DNA present.

We are excited to announce the launch of:

The IvyGene Liver Test is a simple blood test that may confirm the presence of liver cancer as early as stage 1 and provides measurable information about liver cancer. Individuals with cirrhosis, hepatitis, and non-alcoholic fatty liver disease are at a much greater risk for developing liver cancer than the general population. The IvyGene Liver test provides valuable insight into a patient's health at the time of testing.

The IvyGene Liver test is a confirmatory tool meant to be used in conjunction with the normal standards of care. The IvyGene Liver test is approved as a laboratory developed test and is performed at a CLIA registered and CAP accredited laboratory. Screening is not an approved utility of the IvyGene Liver test.

The IvyGene Liver test has a sensitivity of 80% and specificity of 86%.
The IvyGene Liver tests analyzes methylation ratios of specific gene targets in cell-free DNA.

The results of the test are easy to interpret. The results yield a methylation score, which is either between 0-10, which is considered normal and not consistent with the presence of liver cancer or 11-100, which is considered elevated and consistent with the presence of liver cancer. These numbers are based on validations from samples with clinically diagnosed liver cancer, other types of cancer, and healthy patients. These validations include samples drawn from subjects diagnosed with hepatocellular carcinoma and cholangiocarcinoma (bile duct cancers).

Oncogenesis Alters DNA Methylation Patterns

• DNA methylation is an epigenetic regulator of gene expression
• Cytosine (C) may be methylated to form 5-methylcytosine (5mC) when 5’ adjacent to Guanosine (G). These dinucleotide sites are called CpG sites
• Clusters of CpG sites (CpG islands) are found in gene regulatory regions
• Select alterations in DNA methylation consistently occur for some and perhaps all cancers

Methods

Marker Selection, Screening and Validation

• DNA methylation is an epigenetic regulator of gene expression Initial marker screening performed using cfDNA from 1,933 patient samples3
• Final targets were selected and adapted for use on the IvyGene Platform

Table 1. Validation Study Cohort

• All cancer patients were diagnosed according to current medical practice
• All liver cancer patients were diagnosed with hepatocellular carcinoma (HCC)

• The IvyGene Platform consists of methods and procedures for the collection,processing and quantitative analysis of methylation of cell-free DNA
•The IvyGene Dx Liver Cancer Test measures DNA methylation at select CpG sites that highly correlate with hepatocellular carcinoma (HCC)
• Normal, healthy subjects had no symptoms or history of cancer
• Benign liver diseases included patients diagnosed with: cirrhosis (1), HBV (2) benign liver nodule (3) and hepatic cyst (4)

ResultS

Table 2. Test Performance

References:

1. World Health Organization. GLOBOCAN 2018. Graph production: IARC (http://gco.iarc.fr/today)
2. Fast Stats: An interactive tool for access to SEER cancer statistics. Surveillance Research Program, National Cancer Institute. https://seer. cancer.gov/faststats. (Accessed on 11-2-2018)
3. Xu, R. H., et. al. (2017) Circulating tumor DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nature materials 16, 1155-1161.
4.Chou, R., et. al. (2015) Imaging Techniques for the Diagnosis of Hepatocellular Carcinoma: A Systematic Review and Meta-analysis. Annals of internal medicine 162, 697-711

Human Subjects:

This project was approved by the Institutional Review Boards (IRBs) of Sun Yat-sen University Cancer Center, Xijing Hospital, and West China Hospital. Informed consent was obtained from all patients.