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Recent Advances in Liquid Biopsy for Cancer

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Liquid biopsies search for evidence of disease biomarkers in biological fluids, such as blood or cerebrospinal fluid. For cancer, these biomarkers most commonly include tumor-derived circulating tumor cells (CTCs), circulating cell-free nucleic acids, such as circulating tumor DNA (ctDNA), exosomes, proteins, and phosphoproteins. Since liquid biopsies can detect tumor-associated genetic alterations, they can be used to molecularly stratify tumors to guide the most appropriate targeted treatment for precision oncology.

Biomarker detection can occur in either a targeted manner for specific mutations (e.g., by quantitative or digital PCR) or in an untargeted fashion by next-generation sequencing (NGS), which is particularly advantageous for revealing tumor heterogeneity. Therefore, liquid biopsies have generated a lot of enthusiasm because biofluid collection, especially blood, is minimally invasive and could bypass the need for an invasive, needle or surgical tissue biopsy. Additionally, the ease of collection means that repeated longitudinal analysis is possible, for instance to monitor treatment response, development of resistance, and to detect minimal residual disease (MRD) in patients. Liquid biopsies have also been suggested as a possible screening method for early cancer detection if the method could be made sufficiently specific and sensitive to detect low level biomarkers at the start of tumorigenesis.

Early cancer detection: I’ll have the protein-DNA combo please

Cancer is often detected in the later-stages of the disease, when the tumor is larger, and metastasis has occurred, which significantly worsens a patient’s survival prospects. Early cancer detection would help diagnose patients in the beginning stages of the disease when tumors are resectable and the cure rates high. When liquid biopsies emerged, they were hailed as a possible screening mechanism for cancer, but proved at first to lack sufficient specificity and sensitivity for early detection. In two recently published seminal papers, investigators managed to boost the specificity and sensitivity of liquid biopsies for early cancer diagnosis by combining ctDNA with protein detection.

Previously, liquid biopsies either assayed ctDNA or protein. ctDNA is exquisitely specific because somatic mutations are usually linked to tumors, but massively parallel sequencing is too error prone to dependably identify rare mutant ctDNA among abundant regular DNA. “The amount of ctDNA in the blood in early-stage cancer is very low and can constitute only 0.01% of the total cell free DNA,” explained Nickolas Papadopoulos, PhD, Professor of Oncology at the Johns Hopkins Kimmel Cancer Center. “This can create stochastic events based on the amount of blood used for the test. In addition, many cancers do not ‘shed’ their tumor DNA into the circulation, as we’ve previously shown. So, we expected that some cancers would not be detected if only ctDNA were assayed and the test, though specific, would not be sufficiently sensitive.”

On the other hand, protein quantification is highly sensitive although not highly specific. For example, carbohydrate antigen 19-9 (CA 19-9), a pancreatic cancer biomarker, occurs in other diseases or in asymptotic individuals, or may be absent from pancreatic cancer patients. “In order to increase our sensitivity, we needed to add another analyte, so we selected proteins because some are known to be elevated in cancer,” elaborated Dr Papadopoulos. “The issue with protein biomarkers is that they are nonspecific at certain levels. So, we used proteins with high concentration cut off values that are mainly detected in cancer but not in non-cancerous diseases.” The resultant ctDNA and protein combination liquid biopsy proved to have greater specificity and sensitivity for earlier cancer detection.

The first report of dual ctDNA and protein liquid biopsy comprised pancreatic ductal adenocarcinoma (PDAC) patients with early-stage IA, IB, IIA, and IIB cancer, which were compared to a healthy control group. Safe-Sequencing System (Safe-SeqS) was used to detect ctDNA bearing KRAS mutations, the most recurrent mutant PDAC gene, combined with protein quantification of CA 19-9, carcinoembryonic antigen (CEA), hepatocyte growth factor (HGF), and osteopontin (OPN). The analyte panel led to a sensitivity of 64% with a specificity of 99.5%, far greater than for any individual analyte. A high specificity would be indispensable for preventing alarm from false positives in screened healthy individuals.

The dual ctDNA and protein principle was extended in a second report to patients with other early-stage cancers, including ovarian, liver, stomach, pancreatic, esophageal, colorectal, lung, and breast. The test, named CancerSEEK, employed PCR to concurrently detect mutations in the 16 top most mutated genes across the cancer panel combined with quantification of 8 proteins CA19-9, CEA, HGF, OPN, cancer antigen 125 (CA-125), myeloperoxidase (MPO), prolactin (PRL), and tissue inhibitor of metalloproteinases 1 (TIMP-1). CancerSEEK demonstrated a median sensitivity of 70% (lowest for breast cancer at 33%, highest for ovarian cancer at 98%) with a specificity higher than 99% for all tested cancers. Applying a machine learning algorithm could even help locate the tumor, which would be able to be corroborated by conventional tests, such as mammograms or colonoscopy. Will CancerSEEK seek out other cancers? “The test in effect is pan-cancer and we continue on this front,” answered Dr Papadopoulos.

The researchers believe that CancerSEEK could cost less than $500, similar to current screening cancer tests, and that its specificity and sensitivity may be improved by combination with additional tumor biomarkers. “We have initiated prospective studies to test CancerSEEK. This is the way to really evaluate the clinical applicability of the test,” concluded Dr Papadopoulos when discussing the test’s future prospects.

Next-generation sequencing: The solo artist or the orchestra?

Driver mutations are an overriding paradigm in oncology and regarded as the main cause and development of many tumors. Precision oncology frequently seeks targetable driver mutations as a personalized treatment, for example as tyrosine kinase inhibitors (TKIs) against tumors with mutant epidermal growth factor receptor (EGFR). A number of commercial kits have been cleared by the FDA for targeted detection of single driver mutations by liquid biopsy, such as the cobas EGFR Mutation Test v2. This kit detects EGFR exon 19 deletions or exon 21 L858R mutations to identify non-small cell lung carcinoma (NSCLC) patients that should receive targeted erlotinib treatment.

In contrast to the detection of a single genetic alteration, NGS aims to catalog the full scope of genetic alterations present across diverse cancer subtypes, thus refining the molecular stratification of tumors, which can be correlated with optimal treatment selection and patient prognosis. Moreover, NGS can detect several mutations that could be responsible for driving tumorigenesis and identify resistance mechanisms that may have evolved from pre-existing clones after treatment. Since novel, and sometimes rare, actionable mutations are continually being identified and new drugs are under development to target them, the number of mutations that need to be tested per biopsy is rising, making NGS better suited than targeted approaches.

Although initially used on tissue biopsy, researchers are now testing the utility of NGS for liquid biopsy. “Sometimes, tumor tissue is unavailable or inadequate for molecular testing,” explained Professor Nicola Normanno, Director of Translational Research Department of the Istituto Nazionale Tumori in Naples, Italy. “In these instances, ctDNA detection by liquid biopsy is useful, and with the NGS platform, we would be able to obtain a more in-depth molecular characterization of the tumor.” Professor Normanno is addressing some of the challenges of using NGS in liquid biopsy through several pivotal works. In a study of NGS liquid biopsy in metastatic NSCLC and colorectal cancer (CRC) patients, he and his team highlighted several important challenges. “The central point from our study is that we need to work more on specificity and sensitivity to move NGS liquid biopsy into the clinic,” he said of the study. “This is due to the low level of ctDNA in blood samples but also due to potential artifacts detected by NGS which are from clonal hematopoiesis rather than from tumor-associated DNA. So, we need to understand more about the biology of ctDNA in order to improve the technology.”

Tissue biopsies are sampled from a small part of the tumor, which may not be representative of the entire tumor since they are highly heterogenous. “We have ourselves seen a significant level of intratumor heterogeneity, which evolved with treatment, but that observation necessitated multiple biopsies per patients,” Professor Normanno described of a study in CRC patients. “On the other hand, a key advantage of NGS liquid biopsies is that it detects mutations in the blood arising from the entire tumor, overcoming the problem of tumor heterogeneity. It can also be used to monitor treatment response and development of resistance.” When asked about other potential uses of NGS liquid biopsy, Professor Normanno discussed the possibility of evaluating tumor mutational burden (TMB), which is being considered as a potential biomarker for response to immunotherapy. “Right now, there is no clear consensus on how to assess TMB by liquid biopsy. There are a couple of commercial kits in clinical development and clinical trials to understand whether these kits can really recapitulate TMB. I think we still need more time, but this is an active area we are pursuing.”

Will liquid biopsies completely replace tissue biopsies? “I don’t think that will happen, at least not yet. Tissue biopsies are still very useful for other clinical characteristics, so the two types of biopsy can provide complementary information. However, for molecular profiling, NGS liquid biopsy will be relevant for cancer care and we will see a great expansion in its use in the next few years,” Professor Normanno concluded.

A significant amount of progress has been made towards advances in liquid biopsies. Research has demonstrated their potential usefulness for a range of applications, from early cancer detection for diagnosis to molecular characterization of the tumor for treatment optimization and monitoring. Ultimately, randomized controlled trials will be needed to ascertain the ultimate benefit of liquid biopsies in a clinical setting. If they demonstrate benefit, they have the potential to revolutionize clinical oncology practice.