Background: The detection of circulating tumor DNA (ctDNA) during neoadjuvant therapy (NAT) may serve as an early indicator of emerging resistance and disease progression. In this study, we analyzed ctDNA from high-risk early breast cancer patients who received NAT and definitive surgery in the I-SPY 2 TRIAL (NCT01042379). We hypothesized that ctDNA can serve as a biomarker of response and survival in this setting.
Methods: ctDNA analysis was performed on 291 plasma samples from 84 high-risk stage II and III breast cancer patients randomized either to an investigational agent MK-2206, an AKT inhibitor, in combination with paclitaxel followed by doxorubicin and cyclophosphamide (AC) (n=52)—or standard-of-care (paclitaxel followed by AC) (n=32). HER2+ patients also received trastuzumab. Serial plasma was collected at pretreatment (T0), at 3 weeks after initiation of paclitaxel treatment (T1), between paclitaxel and AC regimens (T2), and after NAT prior to surgery (T3).
A personalized ctDNA test was designed to detect a set of 16 patient-specific somatic variants, initially identified from whole exome sequencing of pretreatment tumor, then tested in plasma samples. Regions containing the somatic variants were amplified from cell-free DNA using specific polymerase chain reaction primers. Amplified products were subjected to ultra-deep sequencing (mean: 94,000x) to detect somatic variants. Association between ctDNA and clinicopathologic variables was assessed using Fisher’s exact test. Association of ctDNA with response and survival was analyzed using logistic and Cox regressions, respectively. The survival endpoint of the study was distant disease-free survival. The median follow-up was 4.8 years.
Results: At pretreatment (T0), 61 of the 84 (73%) patients had detectable ctDNA. Pretreatment (T0) ctDNA positivity and levels (mean mutant molecules per mL of plasma) were significantly associated with increased tumor burden (clinical T stage T3/T4), more aggressive tumor biology (higher Mammaprint scores) and subtype (HER2+ and Triple negative). CtDNA detection during NAT decreased over time (T0- 73%; T1- 35%; T2- 14%; T3- 9%).
Of the 84 patients, 23 (27%) achieved pCR and all were ctDNA-negative after NAT (T3), while all 6 patients who had detectable ctDNA at T3 did not achieve pCR. Patients who cleared ctDNA early at T1 (n=27, 48% pCR rate) had significantly increased probability of achieving a pathologic complete response (pCR) compared to those who remained ctDNA-positive (n=29, 17% pCR rate; Odds ratio=4.33, Likelihood ratio p=0.012).
Patients who were ctDNA-positive at T3 (n=6) had significantly increased risk of metastatic recurrence (HR 14.7; 95% CI 1.6-131.5) compared to those who achieved pCR and were ctDNA-negative (n=17). The risk of metastatic recurrence in patients who cleared ctDNA during NAT was not significantly different from those who were negative at T0 and remained negative by T3 (hazard ratio, HR: 2.1, 95% CI: 0.22-20.2). Interestingly, patients who were ctDNA-negative (n=37) but failed to achieve pCR had similar risk of metastatic recurrence with those who achieved pCR (HR 1.4; 95% CI 0.15-13.5).
Conclusions: Early clearance of ctDNA during NAT was significantly associated with increased likelihood of achieving pCR. Residual ctDNA after NAT was a significant predictor of metastatic recurrence, while clearance of ctDNA at any point during NAT was associated with improved outcomes. Taken together, personalized monitoring of ctDNA during NAT may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival. Validation studies in a larger cohort are warranted.
Citation Format: Mark Jesus M Magbanua, Lamorna Brown-Swigart, Gillian Hirst, Christina Yau, Denise Wolf, Hsin-Ta Wu, Antony Tin, Svetlana Shchegrova, Himanshu Sethi, Raheleh Salari, Alexey Aleshin, Maggie Louie, Bernhard Zimmermann, Angela DeMichele, Minetta Liu, Amy Delson, Amy Jo Chien, Smita Asare, Laura Esserman, I-SPY 2 TRIAL Consortium, Laura van't Veer. Personalized monitoring of circulating tumor DNA during neoadjuvant therapy in high-risk early stage breast cancer reflects response and risk of metastatic recurrence [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-04.