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Science in Focus: Circulating Tumour DNA as a Liquid Biopsy

2016; Elsevier BV; Volume: 28; Issue: 12 Linguagem: Inglês

10.1016/j.clon.2016.08.007

1433-2981

Ben O’Leary, Nicholas C. Turner,

Lung Cancer Treatments and Mutations

Circulating nucleic acids in the blood were identified in the 1940s [[1]Mandel P. Metais P. Les acides nucléiques du plasma sanguin chez l'homme.C R Seances Soc Biol Fil. 1948; 142: 241-243PubMed Google Scholar] and observed in the 1970s to fluctuate with treatment in cancer patients [[2]Leon S.A. Sklaroff D.M. Yaros M.J. Free DNA in the serum of cancer patients and the effect of therapy.Cancer Res. 1977; 37: 646-650PubMed Google Scholar]. The human genome was first sequenced using the chain termination method that had won Frederick Sanger his second Nobel prize [3Lander E.S. Linton L.M. Birren B. et al.Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17763) Google Scholar, 4Venter J.C. Adams M.D. Myers E.W. et al.The sequence of the human genome.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10608) Google Scholar]; subsequent rapid developments in sequencing technology alongside reductions in cost have increased clinical applications. Improved accuracy has now made it viable to sequence cell-free DNA in many branches of medicine. The first clinical use of cell-free DNA analysis was in prenatal diagnostics. The identification of trisomies and other genomic disorders, through analysis of copy number differences from foetal DNA in maternal blood, surpasses invasive techniques in accuracy [[5]Lo Y.M.D. Corbetta N. Chamberlain P.F. et al.Presence of fetal DNA in maternal plasma and serum.Lancet. 1997; 350: 485-487Abstract Full Text Full Text PDF PubMed Scopus (2335) Google Scholar]. Substantial clinical advances are being seen in cancer medicine, to elucidate cancer genetic events and the response to treatment from analysis of circulating tumour DNA (ctDNA). Cancers evolve over time, with emergence, expansion and regression of different clones depending on selection pressures. With targeted therapies available, repeat molecular characterisation over time is increasingly important. ctDNA could offer a minimally invasive surrogate for tissue biopsy in this scenario, with serial sampling presenting further opportunities to monitor the emergence of treatment-resistant clones or radiographically occult disease. Cell-free plasma DNA isolated from the blood forms a characteristic pattern of fragment lengths, with peaks approximately corresponding to multiples of the 180 base pair length of DNA bound by a nucleosomal unit [[6]Jahr S. Hentze H. Englisch S. et al.DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.Cancer Res. 2001; 61: 1659-1665PubMed Google Scholar], suggesting that apoptosis with protection of DNA wound around a nucleosome is the major source of plasma DNA. Recent work shows smaller fragments (reflecting DNA bound to transcription factors) spaced throughout the genome that can be used to infer the tissue of origin [[7]Snyder M.W. Kircher M. Hill A.J. Daza R.M. Shendure J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin.Cell. 2016; 164: 57-68Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar]. This confirms that even in many patients with advanced cancer most plasma DNA is derived from white blood cells and not from tumours. Sampling immediately after surgery suggests that plasma DNA has a half-life of a few hours, meaning a sample taken in steady state represents a dynamic equilibrium with high turnover [[8]Diehl F. Schmidt K. Choti M.A. et al.Circulating mutant DNA to assess tumor dynamics.Nat Med. 2008; 14: 985-990Crossref PubMed Scopus (1879) Google Scholar]. EDTA tubes inhibit DNAses that degrade DNA, but plasma must be separated promptly or a specialised preservative tube used, to prevent a large release of genomic DNA from white blood cells diluting tumour content [9Barra G.B. Santa Rita T.H. Vasques JdA. Chianca C.F. Nery L.F.A. Costa S.S.S. EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples.Clin Biochem. 2015; 48: 976-981Crossref PubMed Scopus (97) Google Scholar, 10Kang Q. Henry N.L. Paoletti C. et al.Comparative analysis of circulating tumor DNA stability In K3EDTA, Streck, and CellSave blood collection tubes.Clin Biochem. 2016 Apr 27; (pii: S0009-9120(16)30040-6, http://dx.doi.org/10.1016/j.clinbiochem.2016.03.012 [Epub ahead of print])Google Scholar]. Cell-free DNA can also be present in the cerebrospinal fluid, urine and stool and can increase in a variety of medical conditions and after exercise [[11]Diaz L.A. Bardelli A. Liquid biopsies: genotyping circulating tumor DNA.J Clin Oncol. 2014; 32: 579-586Crossref PubMed Scopus (1537) Google Scholar]. The exact mechanisms determining cell-free DNA release and dynamics remain unknown. The development of multiple partitioning methods such as digital polymerase chain reaction (PCR) and massively parallel sequencing (also known as next generation sequencing or NGS), allow interrogation of plasma DNA. The fraction of plasma DNA derived from tumour varies greatly between patients with cancer, but is often as low as 1% or less [[12]Diehl F. Li M. Dressman D. et al.Detection and quantification of mutations in the plasma of patients with colorectal tumors.Proc Natl Acad Sci USA. 2005; 102: 16368-16373Crossref PubMed Scopus (916) Google Scholar]. This ctDNA can be analysed by error-correcting NGS methods to assay multiple mutations, copy number variation and structural variants, or methylation (Figure 1). Where a more limited number of aberrations are to be assayed, techniques such as digital PCR (adapted as array, droplet or BEAMing) can offer detection as low as 0.01% [[11]Diaz L.A. Bardelli A. Liquid biopsies: genotyping circulating tumor DNA.J Clin Oncol. 2014; 32: 579-586Crossref PubMed Scopus (1537) Google Scholar]. Molecular barcoding of DNA molecules combined with NGS is now showing comparable sensitivity [[13]Newman A.M. Lovejoy A.F. Klass D.M. et al.Integrated digital error suppression for improved detection of circulating tumor DNA.Nat Biotechnol. 2016 May; 34 (http://dx.doi.org/10.1038/nbt.3520 [Epub 2016 Mar 28]): 547-555Crossref PubMed Scopus (615) Google Scholar]. A wealth of evidence exists that ctDNA analysis correlates well with findings from contemporaneous tumour biopsy, although there is variation between case series. A recent series of 98 patients with mixed tumour types found a sensitivity of between 70 and 93% in patients with aberrations in PIK3CA, TP53, EGFR and ERBB2 [[14]Schwaederle M. Husain H. Fanta P.T. et al.Use of liquid biopsies in clinical oncology: pilot experience in 168 patients.Clin Cancer Res. 2016 May 16; (clincanres-0318, http://dx.doi.org/10.1158/1078-0432.CCR-16-0318)Google Scholar]. Similar agreement was found comparing the mutation rates observed in the cell-free DNA of 15 000 patients with advanced cancer to matched, published tissue datasets [[15]Zill O.A. Banks K.C. Nagy R.J. et al.Somatic genomic landscape of over 15,000 patients with advanced-stage cancer from clinical next-generation sequencing analysis of circulating tumor DNA.J Clin Oncol. 2016; 34 (abstract LBA11501)PubMed Google Scholar]. In a phase IV study of Gefitinib in EGFR-mutated lung cancer, ctDNA identified about 65.7% of biopsy-proven EGFR mutations and ctDNA is now Food and Drug Administration approved for selection of Gefitinib therapy [[16]Douillard J.Y. Ostoros G. Cobo M. et al.First-line gefitinib in caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.Br J Cancer. 2014; 110: 55-62Crossref PubMed Scopus (314) Google Scholar]. In few studies does the sensitivity of ctDNA approach 100%, suggesting the existence of tumours that release little DNA in the blood. There is also growing evidence for ctDNA as a biomarker for acquired resistance. In lung cancer, ctDNA has been used to identify the mutations causing resistance to EGFR-targeted therapy, such as EGFR T790M [[17]Zheng D. Ye X. Zhang M.Z. et al.Plasma EGFR T790M ctDNA status is associated with clinical outcome in advanced NSCLC patients with acquired EGFR-TKI resistance.Sci Reports. 2016; 6: 20913Crossref PubMed Scopus (184) Google Scholar]. In endocrine-resistant breast cancer, oestrogen receptor (ESR1) mutations in ctDNA have been shown to correlate with poor prognosis on subsequent aromatase inhibitor, thus showing promise as a predictive biomarker [[18]Fribbens C. O'Leary B. Kilburn L. et al.Plasma ESR1 mutations and the treatment of estrogen receptor–positive advanced breast cancer.J Clin Oncol. 2016 Jun 6; (JCO673061)Google Scholar]. Similarly, in prostate cancer, mutations/gain of AR, the gene encoding the androgen receptor, can be observed to emerge in patients having treatment with Abiraterone [[19]Romanel A. Tandefelt D.G. Conteduca V. et al.Plasma AR and abiraterone-resistant prostate cancer.Sci Transl Med. 2015; 7: 312re10Crossref PubMed Scopus (322) Google Scholar]. Finally, in colorectal cancer, KRAS-mutated clones expand on anti-EGFR treatment, with poorer prognosis on continuation of treatment [[20]Siravegna G. Mussolin B. Buscarino M. et al.Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients.Nat Med. 2015; 21: 795-801Crossref PubMed Scopus (632) Google Scholar]. These observations require confirmation prospectively, with trials integrating ctDNA. It is increasingly clear that ctDNA analysis has the potential to replace tumour biopsy analysis for patients with advanced cancer. Although there is a low false negative rate, optimal use may be in combination with tumour biopsy to extend sampling over time or as a surrogate when tumour biopsy is not possible. Although ctDNA may offer a better representation of the heterogeneity of a cancer, evidence to support this hypothesis is limited to small groups or single cases where investigators have been able to sequence multiple sites of disease and cell-free DNA [[21]Murtaza M. Dawson S.J. Pogrebniak K. et al.Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer.Nat Commun. 2015; 6: 8760Crossref PubMed Scopus (353) Google Scholar]. From these data it seems that most aberrations found in tissue biopsies are represented in the circulation, with a smaller number being private to each. It remains unclear whether certain metastatic sites contribute more or less to the burden of ctDNA, although a lower burden of ctDNA is observed in patients with primary brain tumours or isolated intracranial metastatic disease, suggesting unequal representation of all compartments within the circulation [22Bettegowda C. Sausen M. Leary R.J. et al.Detection of circulating tumor DNA in early- and late-stage human malignancies.Sci Transl Med. 2014; 6: 224ra24Crossref PubMed Scopus (2989) Google Scholar, 23Garcia-Murillas I. Schiavon G. Weigelt B. et al.Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.Sci Transl Med. 2015; 7 (302ra133–302ra133)Crossref Scopus (754) Google Scholar]. Further light will be shed on this in the future with multiple biopsy and autopsy studies. There are strong data showing the potential for ctDNA as a marker for microscopic residual disease/relapse. In a prospective cohort of 55 breast cancer patients receiving neoadjuvant chemotherapy followed by surgery, detectable ctDNA after surgery predicted subsequent metastatic relapse with high accuracy [[23]Garcia-Murillas I. Schiavon G. Weigelt B. et al.Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.Sci Transl Med. 2015; 7 (302ra133–302ra133)Crossref Scopus (754) Google Scholar]. Furthermore, in patients with no detectable ctDNA after surgery, the emergence of detectable ctDNA on serial sampling also predicted relapse. Detection of ctDNA preceded clinical relapse by a median of 7.9 months. Two smaller retrospective cohorts of 20 patients with breast cancer and 11 patients with colorectal cancer treated with curative intent reported similar results [24Reinert T. Schøler L.V. Thomsen R. et al.Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery.Gut. 65 (4), 2016 Apr 1; : 625-634Crossref Scopus (298) Google Scholar, 25Olsson E. Winter C. George A. et al.Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease.EMBO Mol Med. 2015; 7: 1034-1047Crossref PubMed Scopus (324) Google Scholar]. The largest available retrospective cohort comprises 126 patients with diffuse large B cell lymphoma and a median follow-up of 11 years [[26]Roschewski M. Dunleavy K. Pittaluga S. et al.Circulating tumour DNA and CT monitoring in patients with untreated diffuse large B-cell lymphoma: a correlative biomarker study.Lancet Oncol. 2015; 16: 541-549Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar]. In the 107 patients who achieved a complete remission, the investigators observed a shorter median lead time of 3.5 months with a range of 0–200 months, suggestive of wide variation between and within different cancer types. Is early detection of microscopic residual disease clinically relevant? Unlike patients with haematological malignancies, overt metastatic disease from the more prevalent solid tumours is almost always incurable. However, the extended relapse-free and overall survival advantage seen with adjuvant therapy in breast and colon cancers suggest curative treatment for micrometastases is possible, at least in disease naive to systemic therapy. Ultimately this question will require randomised controlled trials with a range of therapies. Although the cost of NGS has fallen over the last decade it remains relatively expensive, with a time frame slow in terms of treatment pathways. Most critically, to date there are very limited data linking ctDNA characteristics to clinical outcomes. The principal challenge, similar to that faced by genomic medicine in general, is to show convincing clinical utility – that integration of ctDNA analysis into existing and future treatment paradigms will improve outcomes for patients. The ability to detect clinically and radiographically occult disease raises the question of whether cell-free DNA may offer an opportunity for early diagnosis through screening. In a series of 4000 women who underwent prenatal diagnostics of their cell-free DNA with massively parallel sequencing, three asymptomatic cancers were diagnosed from observed cancer aberrations in blood [[27]Amant F. Verheecke M. Wlodarska I. et al.Presymptomatic identification of cancers in pregnant women during noninvasive prenatal testing.JAMA Oncol. 2015; 1: 814-819Crossref PubMed Scopus (147) Google Scholar]. With the low burden of ctDNA seen in patients with localised but clinically evident disease, a major challenge will be the development of an assay that meets screening criteria in terms of acceptable cost, sensitivity and specificity. Furthermore, it is currently unclear how a positive result for any given mutation should best be interpreted, not least because mutations can be identified in normal tissues that do not necessarily subsequently develop malignancy [[28]Martincorena I. Roshan A. Gerstung M. et al.High burden and pervasive positive selection of somatic mutations in normal human skin.Science. 2015; 348: 880-886Crossref PubMed Scopus (1015) Google Scholar]. Modern precision therapeutics faces a significant challenge from the temporal and spatial heterogeneity of cancer, with optimal treatment decisions requiring repeat tumour sampling at multiple time points. ctDNA offers a less invasive means to molecularly characterise a patient's disease, in particular where biopsy is difficult or hazardous, and may provide an advantage with regards heterogeneity. Relapse after curative treatment can be detected earlier with ctDNA than clinically or with imaging in a number of malignancies, although it is not yet clear whether this translates into clinical benefit. Ultimately, a rigorous programme of clinical trials is now required to show how ctDNA can best be integrated into clinical practice to augment patient care.