Circulating free DNA in the management of breast cancer

Breast cancer is the most common cancer in women worldwide. Although metastatic breast cancer is currently incurable, there are a number of endocrine, cytotoxic and biological therapies that benefit some patients though determination of tumor burden remains problematic. Treatment response can be assessed by imaging, the serum biomarker cancer antigen 15-3 (CA 15-3) and the FDA approved CellSearch system, which enumerates circulating tumour cells (CTCs). An increase in CA 15-3 levels or a CTC count of ≥5 cells per 7.5 mL blood is associated with poorer prognosis; however, both methods have a sensitivity of only 60% to 70% (1-3) and imaging often fails to rapidly detect changes in tumour burden. There is a need for improved biomarkers with greater sensitivity and specificity to monitor treatment response, help determine the benefit of new and emerging therapies and provide more accurate means for determining prognosis.

Circulating free DNA (cfDNA) first described over 60 years ago (4), has potential as a “liquid biopsy” to monitor cancer in real time. Elevated levels of cfDNA are observed in cancer, particularly in advanced disease, but have also been suggested for the diagnosis of breast (5) and other cancers (6). However, detection of tumour specific alterations in cfDNA [e.g., mutations, loss of heterozygosity (LOH), hypermethylation] has the potential to provide tumour specific markers and has been more widely investigated [reviewed in (7)]. Some studies have suggested that the proportion of cfDNA, which carries tumour specific alterations termed ctDNA, is variable and represents only a small fraction of total cfDNA (8,9). However, there are currently no consensus protocols either for isolation of cfDNA, or for enrichment of this ctDNA, suggested to be largely associated with low molecular weight fractions (10,11), making comparison between different studies and data difficult.

Our group was the first to report emergence of HER2 amplification in cfDNA in patients who were HER2 negative at diagnosis through analysis of cfDNA (12). We also demonstrated whole genome wide analysis of cfDNA using an SNP 6.0 array and reported common tumour-associated copy number variation in cfDNA of 65 breast cancer patients (13). Rapid developments in next generation sequencing have enabled similar genome-wide analysis of cfDNA (14,15) and other recent studies have shown the emergence of acquired resistance to targeted therapies (16,17) through analysis of cfDNA.

The data presented by Dawson et al. (18) extend this developing field through the combined use of digital PCR and targeted deep sequencing (TDS) to assess serial blood samples in 52 patients with metastatic breast cancer while undergoing treatment. Using a combination of a candidate gene approach to screen for somatic mutations in PIK3CA and TP53 and whole genome paired-end sequencing of primary tumour tissue, they identified point mutations and patient specific somatic structural variants, i.e., ctDNA, in 30 of the 52 patients. Results demonstrated a sensitivity of mutant allele detection of 0.1% for digital PCR and 0.14% for TDS. In some patients ctDNA results were discordant with the primary tumour, suggesting tumour evolution and/or emergence of an original minor clone. One critical question to emerge from these data, which will require follow up in new clinical trials, is what level of alteration/mutation in a key driver is sufficient to initiate a switch in disease management? For those patients in whom mutations could not be observed, probably as a result of lack of analysis of all genes, results were inconclusive; the test appears to work well on finding mutations in both the solid tumor and pairing this with the blood sample. Overall, using a modified bootstrap approach Dawson et al. demonstrated improved sensitivity of ctDNA over both CA 15-3 (85% vs. 59%) and CTCs (90% vs. 67%). In 20 of the patients, for whom blood data was available for three or more time points they showed that fluctuations in ctDNA correlated with treatment response observed by imaging. Similar results were also seen for CTC counts and CA 15-3 but again with less sensitivity. Finally, using a Cox proportional hazards model increasing ctDNA levels and CTC counts were both correlated with poorer overall survival (P<0.0001 and P<0.03, respectively).

Much work is still to be done before sensitive mutation analysis of ctDNA, whether by TDS or another method, becomes routine in the clinic, but rapidly accumulating data presented by Dawson et al. and others (12-14,16-18) show the potential of this approach for sensitive and specific serial sampling to provide a “liquid biopsy” of cancer in real time. As our understanding of the genetic heterogeneity of breast and other cancers develops, this will allow for intelligent design of custom assays to survey ctDNA in cfDNA. Alongside this rapidly advancing technology will likely improve in terms of throughput, sensitivity, cost and ease of use. In the not too distant future, ctDNA/cfDNA analysis has the potential to revolutionise the management of common cancers and as we move into the era of personalised medicine but these tests require standardization so they are reliable and reproducible in the same manner as CTC tests. Despite low numbers of patients in this study, in aggregate it appears that ctDNA is reliable in detecting tumor burden but their role in the clinic will take years to establish, so incorporation into prospective clinical trials would be optimal. Whether such tests can replace or adjunct traditional imaging is unclear, and ctDNA/cfDNA is likely to be as heterogenous as the original tumors themselves.

Acknowledgements

With thanks to funders Cancer Research UK, The National Institute for Health Research, The Roy Castle Lung Cancer Foundation. Pancreatic Cancer UK and Hope Against Cancer.

Disclosure: The authors declare no conflict of interest.

References

1. Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol 2007;25:5287-312. [PubMed]
2. Duffy MJ, Evoy D, McDermott EW. CA 15-3: uses and limitation as a biomarker for breast cancer. Clin Chim Acta 2010;411:1869-74. [PubMed]
3. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781-91. [PubMed]
4. Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l’homme. C R Seances Soc Biol Fil 1948;142:241-3. [PubMed]
5. Huang ZH, Li LH, Hua D. Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients. Cancer Lett 2006;243:64-70. [PubMed]
6. Mead R, Duku M, Bhandari P, et al. Circulating tumour markers can define patients with normal colons, benign polyps, and cancers. Br J Cancer 2011;105:239-45. [PubMed]
7. Jung K, Fleischhacker M, Rabien A. Cell-free DNA in the blood as a solid tumor biomarker--a critical appraisal of the literature. Clin Chim Acta 2010;411:1611-24. [PubMed]
8. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 2011;11:426-37. [PubMed]
9. Gormally E, Caboux E, Vineis P, et al. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat Res 2007;635:105-17. [PubMed]
10. Kuhlmann JD, Schwarzenbach H, Wimberger P, et al. LOH at 6q and 10q in fractionated circulating DNA of ovarian cancer patients is predictive for tumor cell spread and overall survival. BMC Cancer 2012;12:325. [PubMed]
11. Schwarzenbach H, Eichelser C, Kropidlowski J, et al. Loss of heterozygosity at tumor suppressor genes detectable on fractionated circulating cell-free tumor DNA as indicator of breast cancer progression. Clin Cancer Res 2012;18:5719-30. [PubMed]
12. Page K, Hava N, Ward B, et al. Detection of HER2 amplification in circulating free DNA in patients with breast cancer. Br J Cancer 2011;104:1342-8. [PubMed]
13. Shaw JA, Page K, Blighe K, et al. Genomic analysis of circulating cell-free DNA infers breast cancer dormancy. Genome Res 2012;22:220-31. [PubMed]
14. Beck J, Urnovitz HB, Mitchell WM, et al. Next generation sequencing of serum circulating nucleic acids from patients with invasive ductal breast cancer reveals differences to healthy and nonmalignant controls. Mol Cancer Res 2010;8:335-42. [PubMed]
15. Leary RJ, Sausen M, Kinde I, et al. Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Sci Transl Med 2012;4:162ra154.
16. Diaz LA Jr, Williams RT, Wu J, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 2012;486:537-40. [PubMed]
17. Misale S, Yaeger R, Hobor S, et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012;486:532-6. [PubMed]
18. Dawson SJ, Tsui DW, Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 2013;368:1199-209. [PubMed]