Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.

BACKGROUND: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence. PATIENTS AND METHODS: Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years. RESULTS: At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). CONCLUSIONS: Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Sequential Cdk1 and Plk1 phosphorylation of protein tyrosine phosphatase 1B promotes mitotic cell death.

Mitotic cell death following prolonged arrest is an important death mechanism that is not completely understood. This study shows that Protein Tyrosine Phosphatase 1B (PTP1B) undergoes phosphorylation during mitotic arrest induced by microtubule-targeting agents (MTAs) in chronic myeloid leukaemia cells. Inhibition of cyclin-dependent kinase 1 (Cdk1) or polo-like kinase 1 (Plk1) during mitosis prevents PTP1B phosphorylation, implicating these kinases in PTP1B phosphorylation. In support of this, Cdk1 and Plk1 co-immunoprecipitate with endogenous PTP1B from mitotic cells. In addition, active recombinant Cdk1-cyclin B1 directly phosphorylates PTP1B at serine 386 in a kinase assay. Recombinant Plk1 phosphorylates PTP1B on serine 286 and 393 in vitro, however, it requires a priming phosphorylation by Cdk1 at serine 386 highlighting a novel co-operation between Cdk1 and Plk1 in the regulation of PTP1B. Furthermore, overexpression of wild-type PTP1B induced mitotic cell death, which is potentiated by MTAs. Moreover, mutation of serine 286 abrogates the cell death induced by PTP1B, whereas mutation of serine 393 does not, highlighting the importance of serine 286 phosphorylation in the execution of mitotic cell death. Finally, phosphorylation on serine 286 enhanced PTP1B phosphatase activity. Collectively, these data reveal that PTP1B activity promotes mitotic cell death and is regulated by the co-operative action of Cdk1 and Plk1 during mitotic arrest.