What is Cancer?

Cancer is fundamentally a disease of tissue growth regulation caused by multiple genetic disorders. In order for a normal cell to transform into a cancer cell, the genes that regulate cell growth and differentiation must be altered specifically known as oncogenes, tumor-suppressor ​ genes, and microRNA genes. 1

These alterations are usually somatic events occurring in somatic cells, although germ-line mutations that occur in sperm or ova can predispose a person to heritable or familial cancer. A single genetic change is rarely sufficient for the development of a malignant tumor. Most evidence points to a multistep process of sequential alterations in several, often many, oncogenes, tumor-suppressor genes or microRNA genes in cancer cells.

What is a Liquid Biopsy?

A liquid biopsy is a pan cancer genomic analysis done on a sample of blood to determine the presence of cancer tumor genes and their specific types of mutation from circulating tumor DNA(ctDNA). ctDNA is free flowing DNA from tumor cells found in the blood of cancer patients.

An analysis is wider than a test which is specific to a few parameters.

Our Liquid Biopsy seeks out ctDNA and not CTCs(Circulating Tumor Cells) . The major reason is that cell free tumor DNA(ctDNA) is more than 100 times more abundant in the bloodstream - than CTCs. In a 10 mL blood sample, you may only find a handful of CTCs at most, whereas with cell free DNA, there are tens to hundreds of genome equivalents that make it easier to test.

Circulating cell-free tumor DNA (ctDNA) consists mainly of 166 base pair double-strand DNA fragments resulting from apoptosis and necrosis leading to release of nuclear DNA into the circulation. These fragments have a short half life in circulation, ranging from 15 minutes to several hours, due to rapid hepatic and renal clearance, thus ctDNA reflects a real-time genomic signature of the tumor.2

1 Croce CM (January 2008). "Oncogenes and cancer". The New England Journal of Medicine. 358 (5): 502–11. doi:10.1056/NEJMra072367. PMID 18234754. ​ ​ ​ ​ ​ ​ ​ ​ https://doi.org/10.1056%2FNEJMra072367 https://www.ncbi.nlm.nih.gov/pubmed/18234754 2 Mino-Kenudson M. Cons: Can liquid biopsy replace tissue biopsy?—the US experience. Transl Lung Cancer Res 2016;5(4):424-427. doi: 10.21037/tlcr.2016.08.01 http://tlcr.amegroups.com/article/view/8889/7791

Our Liquid Biopsy tests across the whole exon of the gene to provide a more comprehensive picture of all the cancer hotspots within a gene.

Our Liquid biopsy tests for all types of genomic alterations: single nucleotide variants (SNVs), copy number variants (CNVs), fusions (re arrangements), and insertions/deletions (indels). -

In addition, our liquid biopsy test is not designed for only a specific type of cancer, but is a pan cancer test.

Our liquid biopsy takes advantage of ONCOMINE - a global unifying bioinformatic resource of genetic information on cancer and related therapies. It’s based on a cancer microarray database and web-based data-mining platform aimed at facilitating discovery from genome-wide expression analyses. Gene sets can be limited to clinically important annotations including secreted, kinase, membrane, and known gene-drug target pairs to facilitate the discovery of novel biomarkers and therapeutic targets.3

Is it a screening or a Diagnostic test?

It is a diagnostic test as it can be used by symptomatic patients to determine the type of cancer. It can also be used to track the mutations of cancer genes as a result of resistance to medication.

It is able to detect the presence of smaller tumours that imaging scans cannot catch. According to the National Center for Biotechnology Information, Translational Lung Cancer Research Journal & U.S. National Library of Medicine, tumors containing ~50 million malignant cells release sufficient DNA for the detection of ctDNA in blood. In contrast, positron emission tomography—computed tomography imaging generally detects tumors measuring no less than 7 to 10 mm in size and containing ~1 billion cells. 4

This highlights it as a form of screening and/or monitoring as it provides the earliest form of cancer detection especially in cases of recurrence due to resistance.

Detection of cancer gene mutations in ctDNA(like EGFR) has an adequate diagnostic accuracy. This is based on a meta-analysis reviewing 20 studies involving 2,012 cases to assess the

3 ONCOMINE: A Cancer Microarray Database and Integrated Data-Mining Platform https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635162/ 4 Ilié M, Hofman P. Pros: Can tissue biopsy be replaced by liquid biopsy? Transl Lung Cancer Res 2016;5(4):420-423. doi: 10.21037/tlcr.2016.08.06 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009092/ diagnostic performance of cfDNA compared with tumor tissue released in 2014 by Luo et al..5 ​ ​ ​ Then followed by one released in 2015 by Qiu et al. that analysed 27 studies involving 3,110 ​ ​ ​ patients.6 Both had an overall sensitivity of 62%, specificity of 96% and diagnostic odds ratio of 38.270.7

In another study, 87 EGFR negative patients (as per tissue biopsy), were found to be EGFR ​ ​ negative using ctDNA NGS(Liquid biopsy) test. The overall accuracy, sensitivity, specificity, ​ ​ ​ ​ and precision of the ctDNA NGS(Liquid biopsy) test was 96.97%, 91.11%, 100%, and 100% respectively.8

On an ethical note early cancer detection does not guarantee that there is relevant therapy to stop or cure it. This makes many doctors not advice for it.

What is the Clinical benefit of this Liquid Biopsy?

Well, you can call a Liquid Biopsy as biopsy free testing. Why? Because it is the first and only - comprehensive non invasive test for tumor sequencing that is currently in clinical use. Mention - biopsy in front of a patient and watch the anxiety flicker across their face. A biopsy means pain, a potential long recovery, and an agonizing wait for results. Liquid Biopsy is the antithesis of a biopsy. It involves no pain, no recovery, and delivers results in 10 days or less. Biopsy free tests - are changing the way physicians look at a patient’s cancer in real time and giving those patients options they have never had access to before.

“Liquid” biopsies will never replace real biopsies, which are irreplaceable sources of information that cannot be obtained by any other means. However, they are already offering all sorts of additional data that could not be obtained in any other way. In patients who cannot be biopsied, or where biopsies do not have enough tissue, “liquid biopsy” offers a means to perform genetic testing for

5 Luo et al.: Diagnostic value of circulating free DNA for the detection of EGFR mutation status in ​ NSCLC: a systematic review and meta-analysis https://www.nature.com/articles/srep06269 6 Qiu M, Wang J, Xu Y, et al. Circulating tumor DNA is effective for the detection of EGFR mutation in ​ non-small cell lung cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev 2015;24:206-12. http://cebp.aacrjournals.org/content/24/1/206 https://www.ncbi.nlm.nih.gov/pubmed/25339418 7 Pérez-Callejo D, Romero A, Provencio M, Torrente M. Liquid biopsy based biomarkers in non-small cell lung cancer for diagnosis and treatment monitoring. Transl Lung Cancer Res 2016;5(5):455-465. doi: 10.21037/tlcr.2016.10.07 http://tlcr.amegroups.com/article/view/10206/8668#B59 8 Validation of liquid biopsy: plasma cell-free DNA testing in clinical management of advanced non-small cell lung cancer https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757203/ targeted therapy. Also, in patients with advanced disease, it is not feasible to obtain biopsies of every metastatic site. But blood reaches both the primary tumor and the metastases, and materials coming from all can be found in a “liquid biopsy”. Finally, unlike “real” biopsies, blood can be repeatedly obtained and used to monitor the course of the disease, including early detection of response and relapse or emergence of resistance to a particular therapy.9

In the article, “Liquid biopsy based biomarkers in non-small cell lung cancer for diagnosis and ​ treatment monitoring”,by Pérez-Callejo et al. reviews the methodologies available for the isolation ​ ​ ​ and analysis of ctDNA, exosomes, tumor-educated platelets and CTCs, then describe the unique information that can be derived from each of these materials and discusses their clinical applications in lung cancer. Their conclusion is clear; liquid biopsies, particularly CTCs and cfDNA, will guide treatment decision, improve the outcome for lung cancer patients and allow early diagnosis of tumors that are not yet visible on imaging.10

Analysis of ctDNA is useful in treatment selection, for response monitoring, and in studying resistance mechanisms.

Genotyping of cfDNA appears to be complementary to tissue genotyping, since it can be performed repeatedly during treatment with TKIs, and it may be able to identify a subpopulation missed in a single biopsy specimen.11

Measurement of ctDNA levels allowed for earlier response assessment compared to radiographic approaches, facilitating personalized cancer therapy.12

The ability to perform serial blood sampling provides a unique mechanism to monitor the course of disease without having to obtain tissue at frequent intervals.13

So in summary of the above the benefits are: 1. Screening and early detection of cancer 2. Determine risk of metastatic relapse (prognosis): Extremely high sensitivity for detection of cancer burden even after curative care before radiological evidence.

9 Molina-Vila MÁ. Liquid biopsy in lung cancer: present and future. Transl Lung Cancer Res 2016;5(5):452-454. doi: 10.21037/tlcr.2016.10.05 http://tlcr.amegroups.com/article/view/10084/8667 10 Molina-Vila MÁ. Liquid biopsy in lung cancer: present and future. Transl Lung Cancer Res 2016;5(5):452-454. doi: 10.21037/tlcr.2016.10.05 http://tlcr.amegroups.com/article/view/10084/8667 11 Mino-Kenudson M. Cons: Can liquid biopsy replace tissue biopsy?—the US experience. Transl Lung Cancer Res 2016;5(4):424-427. doi: 10.21037/tlcr.2016.08.01 http://tlcr.amegroups.com/article/view/8889/7791 12 Newman AM, Bratman SV, To J, et al. An ultrasensitive method for quantitating circulating tumor DNA ​ with broad patient coverage. Nat Med 2014;20:548-54. https://www.ncbi.nlm.nih.gov/pubmed/24705333 https://www.nature.com/articles/nm.3519 13 Liquid Biopsy Applications https://www.healio.com/hematology-oncology/learn-genomics/introduction-to-liquid-biopsies/liquid-biopsy- applications

3. Development of targeted therapies 4. Tumor heterogeneity analysis: Addresses intra-tumor heterogeneity and supplies an adequate reflection of the tumor 5. Real-time monitoring of responses and resistance to therapy: Extremely high sensitivity for detection of cancer burden even aft 6. Stratification and therapeutic decision-making 7. Constitutes a minimally-invasive procedure with high specificity 8. Facilitate therapeutic decision-making14 9. Detection of response to therapy 10. Detection of new driver mutations 11. Quick turnaround time

Does a Liquid biopsy replace the need of a Tissue Biopsy?

The results of liquid biopsies will not necessarily replace tissue biopsies but will be a pivotal additional tool. There is always going to be a role for tissue-based biopsy, as it yields information

14 Calabuig-Fariñas S, Jantus-Lewintre E, Herreros-Pomares A, Camps C. Circulating tumor cells versus circulating tumor DNA in lung cancer—which one will win? Transl Lung Cancer Res 2016;5(5):466-482. doi: 10.21037/ tlcr.2016.10.02 about morphology (including the microenvironment), tumor type, and possible site of origin, all from histology. 15

A liquid biopsy, or blood sample, can provide the genetic landscape of all cancerous lesions (primary and metastases). In addition, it offers the opportunity to systematically monitor cancer disease as the quantification of circulating tumor DNA (ctDNA) has been shown to correlate with tumor burden. 16

What are the associated oncogenes for each of the above common cancers?

15 Ilié M, Hofman P. Rebuttal from Dr. Hofman and Dr. Ilié. Transl Lung Cancer Res 2016;5(4):428-429. doi: 10.21037/tlcr.2016.08.05 16 Dawson SJ, Tsui DW, Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast ​ cancer. N Engl J Med 2013;368:1199-209. https://www.nejm.org/doi/10.1056/NEJMoa1213261 https://www.ncbi.nlm.nih.gov/pubmed/23484797

Which cancer driver variants are on your panel?

DNA/RNA Gene Selected SNV hotspots CNVs Fusions Extra

DNA & AKT1 GNA11 >900 hotspots including: CCND1 ALK MET ​ exon RNA ALK GNAQ CCND2 BRAF 14 skipping APC GNAS EGFR​: ​ T790M, C797S, L858R, Exon CCND3 ERG AR HRAS 19 del CDK4 ETV1 Tumor ARAF IDH1 CDK6 FGFR1 suppressor BRAF IDH2 KRAS/NRAS:​ ​ G12X, G13X, Q61X EGFR FGFR2 genes: CCND1 KIT ERBB2 FGFR3 APC CCND2 KRAS BRAF​: ​ V600E FGFR1 MET FBXW7 CCND3 MAP2K1 FGFR2 NTRK1 PTEN CDK4 MAP2K2 ALK:​ ​ Exon 21-25 FGFR3 NTRK3 TP53 CDK6 MET MET RET CHEK2 MTOR PIK3CA​:​ E545K, H1047R, E542K MYC ROS1 CTNNB1 MYC DDR2 NRAS AKT1:​ ​ E17K EGFR NTRK1 ERBB2 NTRK3 ESR1​: ​ mutations associated with ERBB3 PDGFRA anti- estrogen resistance ERG PIK3CA ESR1 PTEN TP53:​ ​ mutations associated with ETV1 RAF1 loss of function; R175H, FBXW7 RET R273H/C/L FGFR1 ROS1 FGFR2 SF3B1 ERBB2:​ ​ mutations associated with FGFR3 SMAD4 sensitivity to anti-ERBB2 therapies FGFR4 SMO FLT3 TP53 Recurrent deleterious A​ PC mutations (including p.R876*, p.R1114*, p.Q1378*, p.R1450*)

SMAD4:​ ​ R361C/H

CTNNB1:​ ​ S45F, T41A

What are the detection limits:

● SNVs/short indels: a limit of detection (LOD) down to 0.1% allele frequency (AF) can be achieved with a sensitivity of >80% and specificity of >98%* ● TP53 whole-target SNVs/indels: 0.5% AF (looking at all bases within amplicons) ● Fusions and MET exon skipping: LOD down to 1% can be achieved ● CNV targets: detection as low as 1.4-fold change can be achieved

What are the key benefits to Patients & Doctors?

● It’s a comprehensive pan cancer assay that checks for the genetic evidence(driver genes) of common cancers. That way no cancer is missed and solves complex problems of unknown or confusing primary tumor source. ● It is Non Invasive - no surgery required to get a sample specimen to check for cancer. ● It is Affordable - for the information received it is cheaper ● Fast - 3 to 7 days for results after a quick draw of some blood ● Easily repeatable - this makes it easy for doctors to use it to monitor drug response and make decisions on changing therapy ● Captures tumor heterogeneity - cancer is a combination of diseases caused by different mutations in the genes of tumors. Liquid biopsy captures it all enabling the use multiple targeted therapy for cancer. ● Clear targets - it identifies clear targets for Targeted Cancer Therapy which is very effective. ● Sensitive - it captures DNA from smaller tumors than what scans can detect. This allows a patient to change therapy earlier and avoid serious side effects. It also allows early detection of cancer for better treatment outcomes. ● Detects changes in mutations of a patient’s cancer and directs towards a change in therapy earlier than other tests. ● Indicates targeted therapies for each cancerous mutation thus eliminating guesswork. ● Promotes best use of resources in the fight against cancer.