Wu, Hsin-Ta

Abstract Background: We compared the predictive and prognostic value of ctDNA dynamics in high-risk hormone receptor-positive/HER2-negative (HR+/HER2-) and triple negative breast cancer (TNBC) receiving neoadjuvant chemotherapy (NAC) enrolled in the I-SPY 2 trial (NCT01042379). To our knowledge, this is the largest ctDNA study in breast cancer in the neoadjuvant setting. Methods: Blood samples were collected at pre-treatment (T0), during treatment (T1 at 3 weeks, and T2 at 12 weeks) and after NAC (T3 at 24 weeks) from 106 HR+/HER2- and 97 TNBC patients. Plasma samples (n=734) were analyzed using a personalized and tumor-informed mPCR NGS-based ctDNA test (SignateraTM). Patients, all high risk for recurrence by MammaPrint, received paclitaxel-based treatment +/- experimental therapy followed by anthracycline. The median follow-up was 3.0 years (0.5 to 6.5). The predictive and prognostic value of ctDNA dynamics and status at different timepoints were examined. Our analysis is exploratory and does not adjust for other biomarkers. Results: Pretreatment ctDNA positivity (Fisher p<0.0001) and levels (mean tumor molecules/mL, MTM/mL, t test p=0.0062) were significantly higher in TNBC (90.7%, 14.7 MTM/mL) than in high risk HR+/HER2- (66.0%, 5.5 MTM/mL). Early and late ctDNA clearance during treatment (3 and 12 weeks of NAC) was predictive of pathologic complete response (pCR) and residual cancer burden (RCB), class 0-III, in TNBC but not HR+/HER2- (Table). In both subtypes: (1) ctDNA was a significant negative prognostic factor for distant recurrence-free survival (DRFS) at all timepoints (p<0.05) except at pretreatment; (2) all patients who achieved pCR were ctDNA-negative after NAC; (3) among non-responding patients, ctDNA-negativity after NAC was associated with improved DRFS (Table). Conclusions: The predictive value of ctDNA for prediction of pCR and RCB differed between subtypes (HR+/HER2- vs. TNBC), while similar prognostic value was observed. In TNBC, early clearance of ctDNA at 3 weeks was a significant predictor of favorable response to NAC. Compared to patients who were ctDNA-positive after NAC, ctDNA-negative status in both subtypes was associated with improved DRFS even in patients with residual cancer (no pCR or RCB-II/III). These findings could inform on the design of future studies that seek to demonstrate the utility of ctDNA in the curative setting. Predictive and prognostic significance of ctDNA in early breast cancer in the neoadjuvant setting HR+HER2- (n=106) TNBC (n=97) Predictive value for prediction of pCR and RCB Fisher p-value Fisher p-value Early ctDNA clearance (between T0 and T1) and pCR 0.4521 <0.0001 Late ctDNA clearance (between T0 and T2) and pCR 0.8071 0.0004 Early ctDNA clearance (between T0 and T1) and RCB (0-III) 0.1360 <0.0001 Late ctDNA clearance (between T0 and T2) and RCB (0-III) 0.4869 0.0004 Early ctDNA clearance at T1 and pCR rates pCR rate pCR rate ctDNA clearance (ctDNA+ at T0/ctDNA- at T1) 21% 67% Late ctDNA clearance (betweeNo early clearance (ctDNA+ at T0/ctDNA+ at T1) 13% 14% Prognostic value for prediction of DRFS Log rank p-value Log rank p-value ctDNA at T3 and pCR vs no PCR 0.0002 <0.0001 ctDNA at T3 and RCB (0-I vs II-III) 0.0110 <0.0001 Timepoints: T0 - pretreatment; T1 - three weeks after treatment initiation; T2 - at 12 weeks, between paclitaxel-based and anthracycline regimens; T3- after neoadjuvant chemotherapy prior to surgery Citation Format: Mark Jesus Mendoza Magbanua, Lamorna Brown Swigart, Derrick Renner, Svetlana Shchegrova, Gillian L. Hirst, Christina Yau, Denise M. Wolf, Hsin-Ta Wu, Ekaterina Kalashnikova, Amy L. Delson, A. Jo Chien, Debu Tripathy, Smita Asare, Raheleh Salari, Angel Rodriguez, Bernhard Zimmermann, Himanshu Sethi, Alexey Aleshin, Paul Billings, Rita Nanda, Hope S. Rugo, Laura J. Esserman, Minetta C. Liu, Angela DeMichele, Laura van 't Veer. Comparison of the predictive and prognostic significance of circulating tumor DNA in patients with high risk HER2-negative breast cancer receiving neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB111.

Abstract Background: In the I-SPY 2 TRIAL, the addition of P to standard NAC resulted in more than doubling of the pathologic complete response (pCR) rates for both hormone receptor-positive (HR+)/HER2- and triple-negative (TN) early breast cancer (EBC) patients (pts) compared to NAC only (Nanda et al, JAMA Oncol, 2020). At 3 years, distant recurrence-free survival (DRFS) rates in pts with pCR following NAC+P was >95%. We hypothesized that ctDNA can serve as a predictive biomarker of response and survival in pts treated with NAC. Methods: A personalized ctDNA test (Signatera) was performed on 511 serial plasma samples from 138 pts with high-risk HR+/HER2- (n=77) or TN (n=61) stage II/III EBC. Pts received P with paclitaxel (Tx) followed by AC (P arm, n=42) or standard NAC only (n=96), an exploratory subset of pts evaluated for P efficacy. Plasma was collected; pretreatment (T0), 3 weeks after treatment initiation (T1), between Tx+/-P and AC regimens (T2), and prior to surgery (T3). ctDNA was deemed positive with a minimum of 2 of the pt specific tumor mutation fragments detected in cfDNA. Association of ctDNA with response and survival was analyzed using logistic and Cox regressions with pCR and DRFS as endpoints. Median follow-up was 2.8 years. Results: Detection of ctDNA decreased over time (P arm: T0-81%, T1-50%, T2-19%, T3-3%) and NAC only: T0-82%, T1-65%, T2-26%, T3-10%). ctDNA data at T0 and T1 was available for 96% (132/138) of pts in P arm or NAC only (Table). Among ctDNA+ patients at baseline, clearance at T1 was significantly associated with pCR (OR=1.92, ctDNA+/-; OR=0.27, ctDNA+/+; LR p<0.001). This association remained significant after adjustment for HR status and treatment (LR p<0.001) and P arm or NAC only (P: LR p=0.03; NAC: LR p=0.01). ctDNA data at T0, T1, and T2 was available for 86% (118/138) pts. (Table). Among all ctDNA+ pts at baseline, dynamics through T2 was associated with pCR (OR=1.44, ctDNA+/-/-; OR=0.33, ctDNA+/+/-, OR=0.12, ctDNA+/+/+; LR p=0.0011). This association remained significant when adjusted for HR status and treatment (LR p<0.001). Analysis within individual treatments showed significant association for NAC (LR p=0.040) and a non-significant trend in NAC+P (LR p=0.063), likely due to smaller sample size. All pts who achieved pCR were ctDNA- at T3 (n=34). Among those who failed to achieve pCR (n=81), DRFS was significantly better in ctDNA- (n=72/81; 20 in P and 52 in NAC) versus ctDNA+ pts (n=9/81; 1 in P and 8 in NAC) (adjusted HR 0.13; 95% CI 0.05-0.37). Conclusions: These exploratory results align with our previous findings that early clearance of ctDNA during NAC treatment was significantly associated with increased likelihood of achieving pCR. Additionally, we show that ctDNA clearance can be an early surrogate marker for therapy response assessment. Residual ctDNA after neoadjuvant treatment was a significant predictor of metastatic recurrence and death. Personalized monitoring of ctDNA during the course of NAC is feasible and provides information that can be combined with imaging and pathology, and may help to optimize decision making for de-escalation or escalation of therapy. Larger studies are ongoing. ctDNA dynamics and pCRctDNA status at T0 and T1 (n=132)ctDNA status at T0, T1, and T2 (n=118)ctDNA-/-ctDNA+/-ctDNA+/+ctDNA-/-/-ctDNA+/-/-ctDNA+/+/-ctDNA+/+/+Total, n (%)24 (18)28 (21)80 (61)22 (19)24 (20)43 (36)27 (23)pCR, n (%)9 (38)15 (54)11 (14)9 (41)12 (50)8 (19)2 (7)No pCR, n (%)15 (63)13 (46)69 (86)13 (59)12 (50)35 (81)25 (93) Citation Format: Mark Jesus M Magbanua, Denise Wolf, Derrick Renner, Svetlana Shchegrova, Lamorna Brown Swigart, Christina Yau, Gillian Hirst, Hsin-Ta Wu, Ekaterina Kalashnikova, Antony Tin, Amy Delson, Douglas Yee, Angela DeMichele, Raheleh Salari, Angel Rodriguez, Bernhard Zimmermann, Himanshu Sethi, Alexey Aleshin, Paul Billings, Laura Esserman, Minetta Liu, Rita Nanda, Laura van ‘t Veer, I-SPY 2 Investigators. Personalized ctDNA as a predictive biomarker in high-risk early stage breast cancer (EBC) treated with neoadjuvant chemotherapy (NAC) with or without pembrolizumab (P) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-02.

Abstract 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.