CIRCULATING TUMOR DNA (ctDNA) assays are now commercially available for use in lung cancer and melanoma, where they can identify the presence of specific mutations that drive treatment selection. In breast cancer, ctDNA remains more of a research tool, but this is poised to change.
Debu Tripathy, MD
At the 2018 Miami Breast Cancer Conference, Debu Tripathy, MD, Professor and Chair of the Department of Breast Medical Oncology at The University of Texas MD Anderson Cancer Center, Houston, described the potential value of ctDNA in the management of breast cancer, predicting these assays will soon be in the clinician’s hands.1
What ctDNA Assays Can Do
EVEN EARLY-STAGE breast cancer can shed tumor DNA into the blood circulation, where it can be detected, quantified, and analyzed with high sensitivity and specificity using sequence-based techniques such as digital drop polymerase chain reaction (PCR). These methods have been applied to study serial changes that can be correlated with tumor progression, metastasis, and response or resistance to therapies.
“With techniques such as digital drop PCR, we can amplify DNA, and this makes it especially suitable for detecting very low frequencies,” he said. “This has allowed us to look not only at the DNA of a tumor with reasonably good accuracy, but also with ultrasensitivity, even in patients with early-stage breast cancer.”
In brief, ctDNA can be sequenced for mutations and copy number variants, and these mutations may also predict sensitivity to specific therapies. Variant allele frequency over time can be tracked, and it correlates with tumor burden and response. In the setting of drug resistance, ctDNA can elucidate mutational evolution and selection and may be informative about mechanisms of action. It is predicted that ctDNA assays could eventually be used in early detection, monitoring for recurrence, treatment decisions, and discovery, he said.
ESR1 Mutations: Harbingers of Progression?
IN BREAST CANCER, mutations often become apparent upon exposure to treatment, a common one being the estrogen receptor–alpha gene (ESR1), which is rare in primary breast cancer but has been observed frequently after treatment with aromatase inhibitors.
The ESR1 mutation renders the estrogen receptor constitutively active, and hence, insensitive to endocrine therapy but potentially responsive to estrogen receptor downregulators like fulvestrant (Faslodex), which rapidly degrade the receptor, Dr. Tripathy explained.
“Potentially, ESR1 mutations could be harbingers of progression and could be tested in a way to determine whether we need to change therapy.”— Debu Tripathy, MD
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Using digital drop PCR, researchers have analyzed stored plasma from pivotal breast cancer trials to identify mutations and investigate their associations with treatment response and disease progression. For example, in the BOLERO-2 trial, which led to the approval of the mammalian target of rapamycin (mTOR) inhibitor everolimus (Afinitor) plus the aromatase inhibitor exemestane as second-line therapy, ESR1 mutations were rare (1.3%) in the archived specimens, but over time, on treatment, they were present in the form of D538G and Y5375 mutations in 28.4% of patients.2
The presence of both mutations, or only the Y5375 mutation, heralded no benefit from the addition of everolimus over exemestane alone. On the other hand, for patients with the D538G mutation, the combination was very effective (hazard ratio [HR] = 0.34; P < .00006). Median overall survival was 32 months for everolimus plus exemestane in patients lacking either of these mutations, whereas it dropped to 20 to 26 months for those with one of them and to 15 months when both mutations were present.
In a similar analysis of plasma banked in the SOFEA trial of fulvestrant vs exemestane with or without anastrozole, ESR1 mutations were present in 39% of patients.3 In those patients, fulvestrant was more effective, essentially doubling progression-free survival over the aromatase inhibitor. In ESR1 wild-type patients, the drugs conveyed similar benefit.
In PALOMA-3, which evaluated fulvestrant with and without palbociclib (Ibrance), the findings were different.3 Here, the benefit of the combination was clear in both patients with ESR1-mutated breast cancer and those with ESR1 wild-type disease (and also regardless of PIK3CA mutation status). The lack of difference in drug sensitivity could be predicted based on the mechanism of action of the drugs, Dr. Tripathy said.
The same researchers of these retrospective analyses recently reported that, in serial samples from the SOFEA trial, AKT1 mutations increase at the time of disease progression.4 AKT1 is another common mutation in breast cancer and one that is now “actionable.” ESR1 also rose, in varying degrees, and essentially predicted progression as well. Surprisingly, mutations in KRAS genes, not generally thought to be activated in breast cancer, were also seen in patients with disease progression.
“While the patient populations are small, these data suggest that ESR1 mutations are both prognostic and predictive and tell us that the story is complicated,” Dr. Tripathy said. “Potentially, ESR1 mutations could be harbingers of progression and could be tested in a way to determine whether we need to change therapy prior to clinical progression.”
Mutations Could Predict Response to Therapy
SIMILARLY, RESPONSE to treatment might someday be predicted based on targets identified by ctDNA. In BELLE-3, which evaluated the non-isoform selective PI3 kinase inhibitor buparlisib, response was different for mutant vs wild-type PIK3CA.5 In patients with mutations, the addition of buparlisib to fulvestrant more than doubled the median progression-free survival, but in patients with wild-type disease, no additional benefit was shown. This differential response was shown in ctDNA and in tissue; 80% concordance was shown between the two sampling methods.
Such a finding suggests “this might be a test we can use to predict who may respond to drugs that are targeted against the PIK3CA mutation,” Dr. Tripathy said.
Based on changes in mutations across the treatment trajectory, the data suggest ctDNA can also be used for monitoring treatment response, with changes made to treatments accordingly, he added.
THE ctDNA assay has confirmed that within a given tumor there is multifocal clonal evolution, and this can be sampled in real time. Murtaza et al showcased 1 patient with metastatic breast cancer who received 2 lines of targeted therapy over 3 years and multiple biopsies and plasma samples.6 Mutation levels in the plasma samples reflected the clonal hierarchy inferred from the sequencing of tumor biopsies. Serial changes in mutations shown on ctDNA correlated with different treatment responses between metastatic sites.
As Dr. Tripathy explained, “When disease progresses, one can observe multiple mutations in HER2, AKT, and other genes, and they expand under treatment. This is clonal selection and tumor evolution. You can also see this in multiple metastatic sites, where you pick up multiple clones. Certain mutations, perhaps, may predict for recurrence in certain areas of the body.”
Bardia et al identified polyclonal Rb1 mutations by ctDNA in patients who became resistant to a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, with none being present on pretreatment specimens.7 This suggested that these molecular alterations, which lead to functional loss of Rb1, likely emerged under selective pressure from the CDK4/6 inhibitor and potentially conferred therapeutic resistance, as predicted by the known mechanism of action of this class of drugs.
“One can also track early-stage disease with ctDNA, showing that the presence of certain mutations predict for worse outcome,” he said.
A study by Garcia-Murillas et al used ctDNA to monitor for minimal residual disease after neoadjuvant chemotherapy.8 The presence of ctDNA in plasma, after a single time point or during serial plasma sampling, predicted metastatic relapse with high accuracy. Mutation tracking in serial samples, which revealed intratumoral genetic heterogeneity, increased the sensitivity for predicting relapse, with a median lead time of about 8 months over clinical relapse.
The authors concluded that mutation tracking can identify early breast cancer patients at high risk of relapse and reveal genetic events present in minimal residual disease. Such findings might allow for tailored interventions.
“This, again, is a way to track patients, but until we conduct trials showing we can intervene and change the natural history, this technology really won’t help us,” Dr. Tripathy acknowledged. ■
DISCLOSURE: Dr. Tripathy reported no conflicts of interest.
1. Tripathy D: Future clinical and research applications for cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA). Invited lecture at the 2018 Miami Breast Cancer Conference. Presented March 10, 2018.
2. Chandarlapaty S, Chen D, He W, et al: Prevalence of ESR1 mutations in cell-free DNA and outcomes in metastatic breast cancer secondary analysis of the BOLERO-2 clinical trial. JAMA Oncol 2:1310-1315, 2016.
3. Fribbens C, O’Leary B, Kilburn L, et al: Plasma ESR1 mutations and the treatment of estrogen receptor-positive advanced breast cancer. Clin Oncol 34:2961-2968, 2016.
4. Fribbens C, Garcia-Murillas I, Beaney M, et al: Tracking evolution of aromatase inhibitor resistance with circulating tumour DNA analysis in metastatic breast cancer. Ann Oncol 29:145-153, 2018.
5. Di Leo A, Johnston S, Lee KS, et al: Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): A randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol 19:87-100, 2018.
6. Murtaza M, Dawson SJ, Pogrebniak K, et al: Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer. Nat Commun 6:8760, 2015.
7. Condorelli R, Spring L, O’Shaughnessy J, et al: Polyclonal RB1 mutations and acquired resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer. Ann Oncol. December 11, 2017 (early release online).
8. Garcia-Murillas I, Schiavon G, Weigelt B, et al: Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med 7:302ra133, 2015.