Liquid biopsies in cancer clinical trials

One of the most challenging aspects of cancer research is accessing, acquiring, and analyzing oncologic tissue for genomic and pharmacological testing. Traditionally, this responsibility has been the domain of the surgeon, who typically provides the tumor specimens for testing. Minimally invasive techniques have reduced morbidity in cancer patients but have not eliminated the inconvenience and cost to patients, particularly if they require repeated testing. In addition, as cancers progress and metastasize, the ability to biopsy the primary tumor becomes less relevant, as the molecular alterations responsible for progression may lie in the distant sites that can be small and difficult to reach.

Therefore, the discovery and application of blood-based diagnostics and circulating tumor markers have recently gained significant attention in the field of oncology. These so-called liquid biopsies include molecular biomarkers, circulating tumor cells (CTCs), exosomes, and cell-free deoxyribonucleic acid (cfDNA) (see Figure 1). Although traditional biomarkers, such as carbohydrate antigen 19-9 (CA 19-9) and prostate specific antigen (PSA), are easily obtainable from serum, they can be falsely elevated in conditions of inflammation or obstruction, which limits their usefulness for diagnosis and treatment response.

Figure 1. Circulating tumor cells and DNA as liquid biopsies in gastrointestinal cancer

Circulating tumor DNA (ctDNA)

Meanwhile, more precise tools such as cfDNA (DNA fragments freely circulating in the blood) not only signify whether disease is present, but also provide mutational as well as epigenetic information that can lead to personalized treatment strategies. First discovered more than 50 years ago in humans, cell-free circulating tumor DNA (ctDNA) was detected in higher levels in cancer patients than in healthy individuals.¹ It is thought that tumoral cfDNA is derived from cancer cells undergoing apoptosis or rapid turnover and not necessarily from CTC. In addition to serum, cfDNA has been identified in other bodily fluids, including saliva, urine, ascites, breast milk, sputum, cerebrospinal fluid, bile, and stool with varying frequency and stability. It can be detected through various methodologies, ranging from quantitative and digital polymerase chain reaction  techniques such as BEAMing (beads, emulsion, amplification, magnetics) technology for known mutations to genome-wide analysis with next-generation sequencing for large numbers of genes and targeted deep sequencing to examine the entire exome or enrich regions of interest with increased sensitivity.² The fraction of ctDNA within cfDNA can vary from 90 percent to 0.1 percent, depending on tumor biology and burden, host physiology, and oncologic interventions. An estimated 3.3 percent of ctDNA from 3 x 1010 neoplastic cells in a 100g colon cancer is released into the bloodstream daily.³

Figure 2. Tumor Type

ctDNA in advanced cancer

ctDNA has been identified in 75 percent of patients with advanced pancreatic, ovarian, bladder, and esophagastric cancer; however, most cfDNA studies have focused on breast, colon, and lung cancer (see Figure 2). Initial trials, such as the molecular screening for cancer treatment optimization (MOSCATO-01), evaluated the concordance of cfDNA mutational screen of 50 genes with actual tumor DNA in patients with advanced solid tumors undergoing phase 1 trials. A study of 234 patients with tumor DNA that could be matched to their corresponding cfDNA showed a 55 percent sensitivity for detection and that this response varied depending on tumor type, metastatic sites, albumin, and lines of therapy.⁴

An early proof-of-concept pilot study in metastatic breast cancer revealed that ctDNA could be identified in 29/30 women with somatic mutations of PIK3CA and TP53, a higher rate than the tumor marker CA 15-3. Furthermore, variations in ctDNA correlated with treatment response on radiographic imaging.⁵ Tie and colleagues prospectively collected serum from 250 patients with resected stage II colon cancer to determine the correlation of ctDNA to chemotherapy response and recurrence.⁶ Tumor tissue also was sequenced for 15 key genes, and blood was collected every three months during adjuvant therapy for biomarker analysis.

Of 230 patients with complete follow-up, 79 received postoperative chemotherapy, and 34 experienced a recurrence. Patients who had measurable ctDNA after surgery were at increased risk of recurrence; conversely, patients with non-detectable ctDNA had a three-year recurrence-free survival of 90 percent. Interestingly, patients in whom adjuvant therapy converted their ctDNA from high to low levels did not experience a recurrence at 34 months (last follow-up). More recently, ctDNA profiling of the first 100 patients in the tracking of non-small cell lung cancer through evolution of therapy Rx (also known as the TRACERx) study revealed a differential ability to obtain diagnostic information from squamous cell versus adenocarcinoma. Predictors of detection included tumor necrosis, proliferation index, and lymphatic invasion. By longitudinally following these patients, repeated ctDNA analysis predicted patients who recurred, as well as patients who became resistant to adjuvant therapy.⁷

Liquid biopsies through ctDNA may be a promising tool for early diagnosis, prognosis, recurrence, and response to treatment. As an increasing number of surgically treated tumors are being referred for neoadjuvant therapy, the application of this technology to direct or tailor care likely will affect the multidisciplinary management of cancer. Collection and analysis of ctDNA is and should be embedded in ongoing and future prospective clinical trials.

References

1. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 1977;37(3):646-650.
2. Burgener JM, Rostami A, De Carvalho DD, Bratman SV. Cell-free DNA as a post-treatment surveillance strategy: Current status. Semin Oncol. 2017;44(5):330-346.
3. Cree IA, Uttley L, Buckley Woods H, et al. The evidence base for circulating tumour DNA blood-based biomarkers for the early detection of cancer: A systematic mapping review. BMC Cancer. 2017;17(1):697.
4. Jovelet C, Ileana E, Le Deley MC, et al. Circulating cell-free tumor DNA analysis of 50 genes by next-generation sequencing in the prospective MOSCATO trial. Clin Cancer Res. 2016;22(12):2960-2968.
5. Dawson SJ, Tsui DW, Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 2013;368(13):1199-1209.
6. Tie J, Wang Y, Tomasetti C, et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci Transl Med. 2016;8(346):346ra92.
7. Abbosh C, Birkbak NJ, Wilson GA, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017;545(7655):446-451.