Cancer Liquid Biopsy

Cancer Liquid Biopsy

Is it ready for clinic?

The management of cancer relies on a combination of imaging and tissue biopsy for diagnosis, monitoring, and molecular classification-based patient stratification to ensure appropriate treatment. Conventional tissue biopsy harvests tumor samples with invasive procedures, which are often difficult for patients with advanced disease. Given the well-recognized intratumor genetic heterogeneity [1], the biopsy of small tumor fragments does not necessarily represent all the genetic aberrations in the tumor, but sampling the entire tumor in each patient is not realistic. Moreover, tumors evolve all the time from local to advanced disease and by adapting to selective pressure from treatment.

For better monitoring of tumor molecular changes, liquid biopsy is emerging as a more convenient and potentially lower-cost alternative to tissue biopsy. Liquid biopsy detects genetic aberrations in circulating tumor cells (CTCs) and deoxyribonucleic acid (DNA) from apoptotic and necrotic tumor cells. Blood samples are easily accessible and therefore save the cost of time in an operating room. Also, serial blood specimens can be collected at different stages of cancer management, which enables real-time tumor monitoring. This is of particular importance for selecting a personalized treatment, the goal of which is to provide “the right patient with the right drug at the right dose at the right time” [2].

Potential Utilities for Liquid Biopsy

CTCs and circulating tumor DNA (ctDNA) are the major resources of genetic aberrations in liquid biopsy. CTCs can be separated from blood cells by size or through the antibody specific to tumor cell surface antigens, and the number of CTCs can range from five to 50 per 15 milliliters of blood. The detection of ctDNA, which constitutes as little as 0.01% of the total circulating cell-free DNA (cfDNA), is made possible by the emergence of next-generation sequencing (NGS) and digital polymerase chain reaction (dPCR). Over the past decade, the throughput of sequencing technologies has improved by three orders of magnitude, and the cost has lowered from US$1 billion to about US$1,000 per human genome. The dPCR can detect rare ctDNA sequences that are as little as 0.1% of the total cfDNA without the need for reference standard or standard curves. The dramatic advance in detection methods also enables molecular characterization of exosomes [3] and platelets, both of which have been discovered to bear messenger ribonucleic acid (RNA) mutations that are hallmarks of cancer.

The clinical utility of liquid biopsy has been investigated extensively. For example, an increased number of CTCs has been shown to be associated with poor prognosis in metastatic breast cancer patients [4]. Small animal models have been established from CTCs isolated from small-cell lung cancer patients for drug testing and understanding resistance mechanisms [5]. The diagnostic application of ctDNA also was validated in a cohort of over 600 patients with various cancers, and the presence of ctDNA was detected in more than 75% of the patients with advanced disease [10]. In the same study, an 87.2% sensitivity and a 99.2% specificity of ctDNA were reported in detecting KRAS mutations associated with U.S. Food and Drug Administration (FDA)-approved cancer therapy. Siravegna et al. identified genetic alternations in the ctDNA of colorectal cancer patients with primary or acquired resistance to epidermal growth factor receptor (EGFR) blocking treatment [6], indicating the use of ctDNA in dynamic monitoring of response to therapy. Furthermore, Garcia-Murillas et al. showed that mutation tracking in ctDNA can identify early breast cancer patients at high risk of relapse [7]. Finally, a recent study in a 283-patient cohort reported 71% accuracy in identifying the primary tumor location across six different tumor types by detecting RNA alternations in tumor-educated platelets (TEP), suggesting TEP as a potentially powerful tool in pan-cancer screening.

All the accumulating evidence supports the argument that liquid biopsy has great potential in versatile clinical utilities in oncology. Furthermore, due to the easy accessibility of blood specimens, liquid biopsy tests can be conveniently validated clinically, leading to a shorter time from lab discovery to clinical translation. Therefore, liquid biopsy has generated a lot of excitement recently in U.S. investment markets.

Commercially Developed Liquid Biopsy Kits

Since the end of June 2016, liquid biopsy kits have been commercially available from nine companies, as summarized in Table 1. Of them, Roche Molecular Systems’ cobas EGFR Mutation Test v2 is the first and only liquid biopsy test approved by the FDA. The cobas kit is capable of analyzing both plasma ctDNA and formalin-fixed, paraffin-embed tumor tissues. The plasma ctDNA test was approved 1 June 2016 based on a phase III study (NCT01342965) of the chemotherapy medication Tarceva. Plasma and tissue specimens from the same non-small-cell lung cancer (NSCLC) patients enrolled in the clinical trial were compared for agreement. In more than 200 patients, the plasma was also positive for EGFR mutations in 76.7% (70.5%, 81.9%) of tissue-positive specimens and negative in 98.2% (95.4%, 99.3%) of tissue-negative cases. The NSCLC patients with mutations in ctDNA were treated with Tarceva as a first-line treatment. An improved progression-free survival was observed in this cohort compared to those treated with chemotherapy. The FDA has granted priority review for the premarketing application of the cobas test.

Table 1: Commercial Liquid Biopsy Kits in the United States

Kits Manufacturer Technologies Gene Panel Intended Use Price
cobas EGFR Mutation Test v2 Roche Molecular Systems Real-time PCR, plasma ctDNA EGFR exon 19 deletions or exon 21 (L858R) substitution mutation Screening patients with metastatic NSCLC for first-line treatment with the EGFR tyrosine kinase inhibitor Tarceva N/A
FoundationACT Foundation Medicine NGS, plasma ctDNA 62 cancer-related genes Matching patients with targeted therapeutic options relevant to the mutations, for general use of solid tumors US$5,800
Guardant360 Guardant Health NGS, plasma ctDna 70 cancer genes that cover all actionable somatic alterations recommended by the National Comprehensive Cancer Network (NCCN) Matching patients with targeted therapeutic options relevant to the mutations, for general use of solid tumors US$5,400
OncotypeSEQ Liquid Select Genomic Health nGS, plasma ctDna 17 genes that have either been included in NCCN guidelines, associated with sensitivity or resistance to relevant FDA-approved therapies, or established as eligibility criteria for currently enrolling phase II–IV clinical trials Matching patients with targeted therapies, primarily for stage IV lung, breast, colon, and ovarian cancer, melanoma, and gastrointestinal stromal tumors N/A
Target-Selector Biocept Real-time PCR for plasma ctDNA; CELLSEARCH system for plasma CTC identification, isolation, and enumeration: FISH and IHC for CTC molecular characterization EGFRT790M for lung cancer, HER2 for breast cancer* Monitoring treatment resistance to targeted therapy; available for lung and breast cancer Covered by most insurance companies
Trovera Trovagene Proprietary PCR, blood- or urine-derived ctDNA EGFR, KRA S, and BRAF mutations Matching targeted therapy and monitoring treatmentresponse for pancreatic, NSCLC, colorectal, and ovarian cancer and melanoma Accepts all insurance plans
Myriad myRisk Hereditary Cancer Myriad Genetics NGS, blood- or saliva-derived ctDNA 25 genes related to hereditary cancer Risk evaluation for individuals with high risk for hereditary breast, ovarian, gastric, colorectal pancreatic, prostate, and endometrial cancer and melanoma Covered by most insurance companies
CancerIntercept Detect Pathway Genomics PCR and NGS, plasma ctDNA 96 hot spots in nine cancer-driver genes Early detection of colorectal, breast, lung, and ovarian cancers and melanoma US$699 without subscription
CancerIntercept Monitor Pathway Genomics PCR and NGS, plasma ctDNA 96 hot spots in nine cancer-driver genes Monitoring and treatment for colorectal, breast, lung, and ovarian cancers and melanoma US$999 without subscription
ExoDx Lung(ALK) ExosomeDX qPCR, exsomal RNA in blood Five variants of EML4-ALK mutation Matching NSCLC patients with targeted treatment N/A

*Information of the full-panel gene is not available on the Biocept website.

Other treatment selecting kits all identify alternations in a panel of cancer-related genes that either have associated FDAapproved therapies or therapies that are actively enrolling in clinical trials. While not yet cleared by the FDA, all three tests have been extensively validated with clinical specimens and demonstrated excellent accuracy. In a large-scale, multicenter prospective clinical study initiated in July 2015, FoundationACT demonstrated a 97.6–99.9% positive predictive value in 267 cancer patient samples and a ≥95% sensitivity in detecting all four classes of genetic alternations. Moreover, FoundationACT was 100% concordant with its tissue counterproduct from the same manufacturer, FoundationONE. Guardant360 has been studied in over 20,000 clinical specimens. The validation data were published in 2015 with a 99.9999% analytic sensitivity and a comparable clinical sensitivity to that of tissue-derived NGS [8]. OncotypeSEQ also has a >99% per-sample specificity among 73 cfDNA samples from 60 healthy volunteers. Among the three tests, Guardant360 is the most successful in physician adoption, due in part to the commercial launch of FoundationACT and OncotypeSEQ, announced in May and June 2016. Since its commercial introduction in 2014, Guardant360 has been used by most leading cancer centers and more than 1,000 clinics in the United States, and it has successfully helped in selecting appropriate treatment for patients with breast and lung cancer as well as melanoma.

With noninvasive accessibility and a virtually unlimited supply, urine and saliva are even more attractive resources for liquid biopsy. Trovera is the only liquid biopsy kit capable of detecting ctDNA in both blood and urine. While the urine test is currently only recommended for patients with histolytic disorders, these evidences suggested urine-based Trovera tests as a promising tool for cancer monitoring. An upcoming product from ExosomeDX, ExoDx Prostate(IntelliScore), will be the first urine exosomal RNA-based test in the market. This test is designed to aid in determining the treatment plan for prostate cancer patients by predicting disease aggressiveness. ExoDx Prostate(IntelliScore) has been shown to discriminate high-grade prostate cancer from low-grade and benign disease [9].

While urine bears genetic alternations from various types of solid tumors, a urine-based test is probably most relevant to the management of bladder cancer. Bladder cancer DNA and cells are shed directly into urine; therefore, background DNA/RNA contamination from blood cells is low. The anticipated market for a bladder cancer urine test is huge. Bladder cancer recurs in up to 78% of patients and requires life-time surveillance with cystoscopy, making bladder cancer the most expensive human cancer from diagnosis to death. Oncocyte Corporation is developing PanC-Dx products for screening and confirmatory diagnosis of lung, breast, and bladder cancers. GeneXpert from Cepheid, the expert in quantitative real-time PCR, is a strong competitor in urine-based bladder cancer diagnosis. Unlike other liquid biopsy platforms, GeneXpert is a point-of-care device with an automated sample preparation that provides results in 90 minutes.

Early screening and detection of cancer in asymptomatic individuals has always been the Holy Grail for cancer research. Myriad Genetics’ myRisk Hereditary Cancer and CancerIntercept Detect screens high-risk populations for the presence of genomic alternations in plasma ctDNA as early marks of cancer. Examples of high-risk populations include individuals with significant family history or a previous history of cancer. A simple and well-designed pretest quiz is provided to identify potential candidates for testing. Illumina, the big player in NGS, is also entering the liquid biopsy market. In January 2016, Illumina formed a new company, GRAIL, to develop a pan-cancer blood screening test for ctDNA in asymptomatic individuals. In February 2016, Illumina acquired an Amsterdam-based company, thromboDx, that specializes in detecting tumor RNA markers in blood platelets to augment GRAIL’s early cancer detection.

Looking to the Future

The overall impression is that liquid biopsy is the most promising among current cancer monitoring and guidance treatment selections. However, important questions still need to be answered before liquid biopsy can be used as a common approach in clinics. Fundamentally, does the information provided by liquid biopsy improve patient outcomes? While metastatic breast cancer patients with increased CTCs had an early switch from first-line chemotherapy to second- and third-line treatment, the early switch did not effectively improve the overall survival of such patients. While the disappointing results may be explained by the ineffectiveness of second- and third-line therapy, then how will physicians manage those patients? This is especially important for cancer diagnostic and screening kits.

As a more sensitive approach, liquid biopsy can detect cancer at a much smaller tumor load than imaging technologies. A positive liquid biopsy result that could not be confirmed by definitive imaging is going to raise a lot of patient anxiety, and overtreatment may do harm to the patient. More studies with long-term follow-up are required to demonstrate a positive diagnostic liquid test result as a starting point to initiate treatment. On the other hand, the sensitivity and accuracy of the NGS techniques must be improved for ctDNA detection in localized cancer. But overall, with accumulating clinical evidence, it is just a matter of time until liquid biopsy will replace tissue biopsy.

References

  1. A. Marusyk, V. Almendro, and K. Polyak, “Intra-tumour heterogeneity: A looking glass for cancer?” Nat. Rev. Cancer, vol. 12, no. 5, pp. 323–334, May 2012.
  2. W. Sadée and Z. Dai, “Pharmacogenetics/genomics and personalized medicine,” Hum. Mol. Genet., vol. 14, no. 2, pp. R207–14, Oct. 2005.
  3. J. G. Lohr, V. A. Adalsteinsson, K. Cibulskis, A. D. Choudhury, M. Rosenberg, P. Cruz-Gordillo, J. M. Francis, C. Z. Zhang, A. K. Shalek, R. Satija, J. J. Trombetta, D. Lu, N. Tallapragada, N. Tahirova, S. Kim, B. Blumenstiel, C. Sougnez, A. Lowe, B. Wong, D. Auclair, E. M. Van Allen, M. Nakabayashi, R. T. Lis, G. M. Lee, T. Li, M. S. Chabot, A. Ly, M. Taplin, T. E. Clancy, M. Loda, A. Regev, M. Meyerson, W. C. Hahn, P. W. Kantoff, T. R. Golub, G. Getz, J. S. Boehm, and J. C. Love, “Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer,” Nat. Biotechnol., vol. 32, no. 5, pp. 479–484, 2014.
  4. M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. M. M. Terstappen, and D. F. Hayes, “Circulating tumor cells, disease progression, and survival in metastatic breast cancer,” New Eng. J. Med., vol. 351, no. 8, pp. 781–791, Aug. 2004.
  5. C. L. Hodgkinson, C. J. Morrow, Y. Li, R. L. Metcalf, D. G. Rothwell, F. Trapani, R. Polanski, D. J. Burt, K. L. Simpson, K. Morris, S. D. Pepper, D. Nonaka, A. Greystoke, P. Kelly, B. Bola, M. G. Krebs, J. Antonello, M. Ayub, S. Faulkner, L. Priest, L. Carter, C. Tate, C. J. Miller, F. Blackhall, G. Brady, and C. Dive, “Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer,” Nature Med., vol. 20, no. 8, pp. 897–903, 2014.
  6. G. Siravegna, B. Mussolin, M. Buscarino, G. Corti, A. Cassingena, G. Crisafulli, A. Ponzetti, C. Cremolini, A. Amatu, C. Lauricella, S. Lamba, S. Hobor, A. Avallone, E. Valtorta, G. Rospo, E. Medico, V. Motta, C. Antoniotti, F. Tatangelo, B. Bellosillo, S. Veronese, A. Budillon, C. Montagut, P. Racca, S. Marsoni, A. Falcone, R. B. Corcoran, F. Di Nicolantonio, F. Loupakis, S. Siena, A. Sartore- Bianchi, and A. Bardelli, “Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients,” Nature Med., vol. 21, no. 7, pp. 795–801, 2015.
  7. I. Garcia-Murillas, G. Schiavon, B. Weigelt, C. Ng, S. Hrebien, R. J. Cutts, M. Cheang, P. Osin, A. Nerurkar, I. Kozarewa1, J. Armisen Garrido, M. Dowsett, J. S. Reis-Filho, I. E. Smith, and N. C. Turner, “Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer,” Sci. Translational Med., vol. 7, no. 302, p. 302ra133, Aug. 2015.
  8. R. B. Lanman, S. A. Mortimer, O. A. Zill, D. Sebisanovic, R. Lopez, S. Blau, E. A. Collisson, S. G. Divers, D. S. B. Hoon, E. S. Kopetz, J. Lee, P. G. Nikolinakos, A. M. Baca, B. G. Kermani, H. Eltoukhy, and A. A. Talasaz, “Analytical and clinical validation of a digital sequencing panel for quantitative, highly accurate evaluation of cell-free circulating tumor DNA,” PLoS One, vol. 10, no. 10, p. e0140712, Oct. 2015.
  9. J. McKiernan, M. J. Donovan, V. O’Neill, S. Bentink, M. Noerholm, S. Belzer, J. Skog, M. W. Kattan, A. Partin, G. Andriole, G. Brown, J. T. Wei, I. M. Thompson, Jr., and P. Carroll, “A novel urine exosome gene expression assay to predict high-grade prostate cancer at initial biopsy,” JAMA Oncol., vol. 2, no. 7, pp. 882–889, 2016.
  10. C. Bettegowda, M. Sausen, R. J. Leary, I. Kinde, Y. Wang, N. Agrawal, B. R. Bartlett, H. Wang, B. Luber, R. M. Alani, E. S. Antonarakis, N. S. Azad, A. Bardelli, H. Brem, J. L. Cameron, C. C. Lee, L. A. Fecher, G. L. Gallia, P. Gibbs, D. Le, R. L. Giuntoli, M. Goggins, M. D. Hogarty, M. Holdhoff, S.-M. Hong, Y. Jiao, H. H. Juhl, J. J. Kim, G. Siravegna, D. A. Laheru, C. Lauricella, M. Lim, E. J. Lipson, S. K. N. Marie, G. J. Netto, K. S. Oliner, A. Olivi, L. Olsson, G. J. Riggins, A. Sartore-Bianchi, K. Schmidt, L. Shih, S. M. Oba-Shinjo, S. Siena, D. Theodorescu, J. Tie, T. T. Harkins, S. Veronese, T. Wang, J. D. Weingart, C. L. Wofgang, L. D. Wood, D. Xing, R. H. Hruban, J. Wu, P. J. Allen, C. M. Schmidt, M. A. Choti, V. E. Velculescu, K. W. Kinzler, B. Vogelstein, N. Papadopoulos, and L. A. Diaz, Jr., “Detection of circulating tumor DNA in early- and late-stage human malignancies,” Sci. Translational Med., vol. 6, no. 224, p. 224ra24, Feb. 2014.