Advances in Liquid Biopsies: Improving Sensitivity and Earlier Detection

Cancer is most treatable in the early stages of the disease, and a biopsy is considered the gold standard in diagnosis. It involves directly sampling the suspected tumor by removing a small piece of tissue or a few cells and testing them in the lab to confirm if they are cancerous. But it is invasive and can be painful for the patient.¹

The less painful and minimally invasive alternative is a liquid biopsy. This involves drawing a blood sample and separating it into blood cells and plasma. It is then run through a sequence of tests that look for circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Their presence can be used as evidence of a tumor, and to determine the type of cancer and what treatment may work best.^1,2

Liquid biopsies may be offered if the patient has metastatic cancer or if treatments are no longer working. They can help determine prognosis and make treatment decisions, and are helpful if the tumor is hard to reach or the patient isn’t well enough for a traditional biopsy.¹

Liquid biopsies are an active area of research and scientists are working to develop platforms that can catch cancer early, or even predict cancer before a tumor has formed. This article will focus on some of the advances currently being made in the field, from attempts to improve the sensitivity of tests, to using bodily fluids other than blood.

Overcoming the sensitivity challenge

Retrieve More Information From Limited DNA

Discover how to obtain multi-modal information from liquid biopsies.
View Poster

Advertisement

Liquid biopsies work by detecting ctDNA or CTCs circulating in the bloodstream, but these only occur in small amounts and for the briefest of time before they are cleared by the body, so it is challenging to develop tests sensitive enough to detect them.²

“Diagnostics that use liquid biopsies require detecting very minute quantities of biological molecules associated with the disease or progression,” explained Professor J. Christopher Love, the Raymond A. and Helen E. St. Laurent Professor of Chemical Engineering at Massachusetts Institute of Technology (MIT), and a member of the Koch Institute and the Ragon Institute of MGH, MIT and Harvard.

“One example is cell-free DNA…Most cell-free DNA is released from dying cells and is degraded naturally by multiple mechanisms, including cellular uptake by liver cells and digestion by DNA nucleases. A small portion of this DNA can originate from tumors (usually ~ 1% or less); this DNA may contain mutations or variations that indicate the presence of cancer. One challenge for these tests is that the amount of tumor-derived DNA in one or two tubes of blood may be too small to detect reliably.”

Most efforts to improve the sensitivity of liquid biopsies tend to focus on developing new sequencing technologies to use after the blood is drawn.² Professor Love and colleagues from MIT and the Broad Institute of MIT and Harvard have developed a way to significantly enhance the sensitivity of liquid biopsies by slowing down the clearance of tumor DNA from the bloodstream, before blood is drawn.

“Our work has explored a new idea similar to contrast agents used in other diagnostics like ultrasound and MRI. Our labs have developed two different ways to temporarily slow down the degradation of the cell-free DNA, and by doing so, boost the amount of DNA available in a tube of blood by 10–60x to make detection more robust,” Love said.

“One approach uses a form of 'dummy' nanoparticles that can distract the liver from collecting cell-free DNA in circulation. The other uses a monoclonal antibody, similar to many biopharmaceuticals used today for treating cancer, to bind and protect the cell-free DNA from degradation temporarily.”

The joint study focused specifically on lung cancer and mouse models has shown that the two priming agents can boost DNA levels enough that the percentage of detectable early-stage lung metastases leapt from less than 10% to over 75%.³

“We are excited that this concept for priming agents could help enhance the benefits of blood-based cancer diagnostics,” added Love.

“Boosting the levels of cell-free DNA prior to a blood draw could improve the ability to detect recurrence of disease, improve the selection of therapies for cancer patients, and ultimately, early detection of cancer.”

A key next step, Love explained, is to understand if this effect can be seen in humans and if this approach can be safe for patients in clinical trials, but with greater sensitivity, liquid biopsies could become more useful for far more patients.

Looking for dark matter

Daniel Kim, assistant professor of biomolecular engineering at the University of California Santa Cruz has taken a different approach to diagnosing cancer with liquid biopsies; his laboratory has been honing in on signals from “dark matter” RNA.

“RNA dark matter is RNA that does not encode for proteins, which is why we refer to it as noncoding RNA,” explained Kim. “While the purpose of most RNA dark matter is unclear, we have found that many noncoding RNAs are aberrantly upregulated at the earliest stages of cancer and preferentially secreted from cancer cells.”

Around 75% of the human genome generates noncoding RNA, many of which are derived from repetitive elements. There would be few of these repetitive noncoding RNAs in a healthy individual’s blood, but Kim has shown that they are expelled from cancer cells, even early in the disease. Each type of cancer has its own cell-free RNA profile, with many RNAs coming from repeated elements.^4,5

Kim’s team developed an RNA liquid biopsy platform that detects RNA dark matter “by taking a biofluid sample such as blood, isolating cell-free RNA, sequencing the RNA, and performing computational analysis to identify and quantify the RNA dark matter, which can tell us whether a patient has cancer,” Kim said.

“RNA dark matter is highly abundant in the blood of cancer patients, even at the earliest stages, which enables us to significantly improve the diagnosis of early-stage cancers, which DNA-based liquid biopsies often miss.”

Kim’s team are now developing a highly sensitive and specific RNA liquid biopsy test for multi-cancer early detection.

Clues from other bodily fluids

Other bodily fluids besides blood also offer useful information about the presence of cancer; urine has been shown to harbor markers of bladder cancer, while saliva and lymphatic fluid collected after surgery can be indicative of head and neck cancers.⁶

Breast milk from cancer patients could also be used as an early diagnostic tool during pregnancy and in the postpartum period, according to researchers at the Vall d’Hebron University Hospital Breast Unit who found breast milk contained ctDNA with the same mutations that present in the primary tumor.^7,8

“From there we have designed an agnostic gene panel containing the mutations that appear most frequently in women diagnosed with breast cancer before the age of 45,” explained Dr. Cristina Saura, head of the Breast Cancer Unit at Vall d’Hebron University Hospital and Vall d’Hebron Institute of Oncology.

Saura and her colleagues collected blood and breast milk samples from patients and found “the sensitivity of liquid biopsy in blood was only 8%, while in breast milk, if we use ddPCR (droplet digital PCR) it was 87%, and using the agnostic gene panel it was 71%,” Saura said.

“Therefore, there is a clear difference in sensitivity depending on the fluid used for liquid biopsy which we think is related to the amount of ctDNA present in the fluids and which is much higher in breast milk simply because of the contiguity with the tumor.”

The team hope to apply their gene panel in a healthy population to define its sensitivity and specificity, and therefore its usefulness for early detection of breast cancer. If it proves so, Saura said it could be “tremendously useful because it would be aimed at a population for which we do not have any screening tool.

“We also know that postpartum breast cancer has a worse prognosis related in part to biology and immunosuppression that occurs with the physiological involution of the breast after breastfeeding. We know that we will not be able to change that fact, but it would be possible to diagnose these tumors earlier and with early detection we know that we will increase the cure rate by treating patients earlier.”

Post-diagnosis benefits

Liquid biopsies can do so much more than aid cancer diagnosis: they may be a useful tool in precision medicine to show whether the patient is a good candidate for certain types of tailored, targeted treatment.

Measuring CTCs is considered a promising approach to guiding immunotherapy. CTCs are an early marker of the immune response and could be used to guide therapy where traditional imaging struggles to reflect how well the body is responding to treatment. Liquid biopsies may also show which patients may benefit from the available immunotherapies or whether they may develop immune-related side effects.⁹

Liquid biopsies can be drawn periodically to help monitor a patient’s tumor and how their disease is progressing, to provide information on mutations that may be driving resistance to current therapy and adjust accordingly. They may also make it possible to respond to cancer quicker by identifying genetic markers of other cancers before diagnosis with conventional screening.^1,10

But the scope of liquid biopsies stretches much further than cancer. “There is increased recognition that liquid biopsies can reduce the risk of more invasive procedures in prenatal care, neurodegenerative diseases, in addition to cancer,” said Love. “The approach we have developed could also benefit these applications and could also apply to other molecular analytes like proteins or RNA too.”

Liquid biopsies have many benefits and can be used alongside the gold standard of cancer diagnosis to provide healthcare providers with masses of invaluable information about the cancer. Advances can only improve the amount of information available and enable clinicians to follow the correct treatment path to ensure the best outcome, all while being less invasive for the patient.

About the interviewees:

Professor J. Christopher Love is the Raymond A. and Helen E. St. Laurent Professor of Chemical Engineering at Massachusetts Institute of Technology (MIT), and a member of the Koch Institute and the Ragon Institute of MGH, MIT and Harvard. His research interests include micro/nanofabrication and surface chemistries, cellular immunology and infectious diseases, immunotherapy/vaccines andclinical diagnostics.

Daniel Kim is an assistant professor in biomolecular engineering at the University of California Santa Cruz, a visiting assistant professor at Stanford University, an associate member of the Canary Center at Stanford for Cancer Early Detection, and a research scholar of the American Cancer Society. His laboratory develops RNA technologies for precision health, cancer early detection and RNA medicine. His RNA research has been recognized by awards from the Damon Runyon Cancer Research Foundation and the US National Academy of Sciences.

Dr. Cristina Saura is head of the Breast Cancer Unit at Vall d’Hebron University Hospital and group leader of VHIO´s Breast Cancer & Melanoma Group. Her work focusses on breast cancer and she is currently involved in different international studies with drugs targeted against molecular targets in the disease.