ctDNA analysis offers a non-invasive approach for monitoring response and resistance to treatment. Serial ctDNA testing during neoadjuvant therapy (NAT) may provide early indicators 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 hypothesize that (1) assessment of ctDNA levels early in treatment will improve the performance of molecular and imaging-based predictors of pathologic complete response (pCR) to NAT; and (2) levels of ctDNA after NAT are associated with residual cancer burden and recurrence [distant recurrence free survival (DRFS)]. Methods: ctDNA analysis was performed in 84 high-risk stage II and III breast cancer patients randomized to neoadjuvant investigational agent (n=52), AKT inhibitor MK-2206 (M) in combination with paclitaxel (T) followed by doxorubicin and cyclophosphamide (AC) (M+T->AC), or standard-of-care (T->AC) (n=32). HER2+ patients also received trastuzumab (H). Serial plasma was collected before NAT (T0), early treatment (3 weeks, T1), between regimens (12 weeks, T2), and after NAT prior to surgery (T3). Mutational profiles derived from pretreatment tumor biopsy and normal matched DNA whole exome sequencing were used to design personalized assays targeting 16 patient-specific somatic variants to detect ctDNA in serial plasma. Results: Of the 84 patients in this study, 43% were HR-/HER2- (TNBC), 35% HR+/HER2-, and 23% HER2+. In total, 74% (61 of 83), 35% (28 of 79), 14% (9 of 65), and 8% (5 of 61) were positive for ctDNA at timepoints T0, T1, T2, and T3, respectively. At T0, ctDNA positivity and levels (average number of mutant molecules detected per mL) were significantly associated with increased tumor burden (by clinical and MRI examination), more aggressive tumor biology (as reflected in higher Mammaprint scores and grade) and subtype (HER2+ and TNBC). Twenty-seven percent (27%) of the 84 patients achieved a pCR and all patients who were ctDNA-positive at T3 (n=5) did not achieve a pCR. Currently, data are being collected to: (1) assess the relationship of ctDNA and MRI imaging in predicting tumor response to therapy; (2) examine the relationship of ctDNA levels before and after NAT with 3-year DRFS and event-free survival (EFS). The results of these analyses will be presented at the SABCS 2018 meeting. Conclusions: Our study provides a platform to evaluate the clinical significance of ctDNA for serial monitoring of response to NAT. Accurate and early response prediction by highly sensitive ctDNA analysis can facilitate a timely and judicious change in treatment to improve patients' chances of achieving a pCR. Finally, personalized ctDNA testing may complement imaging and pathologic evaluation of tumor response to fine-tune pCR as a surrogate endpoint for improved DRFS and EFS. Citation Format: Magbanua MJM, Brown-Swigart L, Hirst GL, Yau C, Wolf D, Ma AA, Bergin E, Venters S, Sethi H, Wu H-T, Salari R, Tin T, Sawyer S, Louie M, Zimmermann B, Lin C-HJ, Keats J, Liang WS, Cuyugan L, Enriquez D, Tripathy D, Chien AJ, Forero A, DeMichele A, Liu M, Delson AL, Asare S, Esserman L, van't Veer L, I-SPY 2 Consortium. Personalized serial circulating tumor DNA (ctDNA) analysis in high-risk early stage breast cancer patients to monitor and predict response to neoadjuvant therapy and outcome in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-01.

Background: Clinical studies have demonstrated the prognostic and predictive value of the number of CTCs in metastatic breast cancer. However, it is the molecular characterization of CTCs that offers insight into the biology of these tumor cells in the context of personalized treatment. Methods: Ten to twenty mLs of blood from 32 metastatic breast cancer patients with poor prognosis (≥5 CTCs in 7.5mLs blood) were subjected to immunomagnetic enrichment. Pools of ∼20 CTCs [EpCAM+, CD45-, nucleated] were isolated via fluorescence activated cell sorting, FACS. Genomic DNA was subjected to whole genome amplification followed by array comparative genomic hybridization (CGH) analysis. Serial genomic profiling was performed in three patients. Genomic profiles from archival primary tumor available from 4 patients were compared to matched CTCs. Copy number analysis was performed using the Nexus® Copy Number. Genomic alterations with p ≥0.05 were considered significant. Results: Genomic profiling of CTCs revealed significant recurrent (≥35%) genomic alterations including gains in 8q12.1, 8q24 and losses in 1p36, 4p16, 5q21, 10q22, 11q24-25, and 13q34. Good concordance of CGH profiles obtained from CTCs isolated from serial blood samples attested to the reproducibility of the assay. Furthermore, comparisons of CTCs with matched archival primary tumors confirmed shared lineage with some divergence. Conclusions: Copy number analysis revealed common genomic alterations in CTCs as well as shared aberrations with matched primary tumor. In addition, we demonstrated the feasibility of serial genomic profiling of CTCs. Future work will determine associations between genomic aberrations in CTCs and clinicopathological parameters. Uncovering genomic alterations in CTCs may lead to the discovery of therapeutic biomarkers to specifically target these cells in breast cancer patients who respond poorly to current aggressive therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-10.

It is hypothesized that cancer prognosis may be related to the functional status of the immune system. We examined the correlation between peripheral blood CD4/CD8 ratio measured at the time of surgery and clinical outcome in patients diagnosed with early stage breast cancer. Patient and Methods Peripheral blood from 57 treatment-naïve early breast cancer patients, not eligible for neoadjuvant chemotherapy, was collected on the day of definitive surgery. CD4+ and CD8+ T cells were enumerated using flow cytometry and the ratio between the two immune cell populations was calculated. Cox regression analyses were performed to determine the relationship between CD4/CD8 ratio vs. distant disease-free survival (DRFS), breast cancer-specific survival (BCSS) and overall survival (OS). The median follow-up times were 10.1 years (range: 0.4-17.5) and 15.0 (range: 1.0-18.5) for DRFS and BCSS/OS, respectively. Results The patients' mean age at diagnosis was 54 years old (range: 31-78). 82% were hormone receptor-positive, 21% HER2-positive, and 61% node-negative. The median CD4/CD8 ratio was 2; and a ratio ≤ 2 was considered low. CD4/CD8 ratio was not associated with any of the clinicopathologic variable examined. Multivariate analysis using a survival model that adjusted for potential confounding factors (age, tumor size, grade, stage, hormone receptor, HER2, lymph-node status) revealed that patients with low CD4/CD8 ratio have statistically significant increased risk of distant recurrence (DRFS HR 5.3, Wald p=0.0381) and death (OS HR 3.8 Wald p=0.0271). Conclusions Immune dysfunction at the time surgery is correlated with long-term increased risk for metastatic recurrence and death. Larger clinical studies are warranted to confirm the results of this study. Citation Format: Magbanua MJM, Yau C, Scott JH, van't Veer L, Park JW, Esserman L, Campbell M. Low peripheral blood CD4/CD8 ratio at the time of surgery is a negative long-term prognostic factor in women with early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-12.

We examined the prognostic impact of CTCs and DTCs detected at the time of definitive surgery in pts diagnosed with early breast cancer (EBC). Methods: Blood and bone marrow samples from 742 treatment-naïve EBC pts, not eligible for neoadjuvant therapy, were collected immediately prior to surgery. 87% were hormone receptor (HR)-positive, and 71% were node-negative. DTCs (n=584) were enumerated using an EPCAM-based method involving immunomagnetic enrichment and flow cytometry (IE/FC). CTCs were enumerated either by IE/FC (n=288) or CellSearch (n=380). Optimal cutoffs for CTC-/DTC-positivity were selected using Monte-Carlo cross validation. Multivariate Cox regression analysis was performed to determine correlation between levels of CTCs/DTCs vs. distant recurrence-free survival (DRFS) and breast cancer-specific survival (BCSS). The overall median follow-up was 7.1 years for DRFS and and 9.1 years for BCSS, but extended up to 13.3 years in subset analyses (Table 1). Results: CTC-positivity by CellSearch was associated with HER2-positivity (Fisher p=0.01). Using optimized cutoffs in multivariate analyses, we found that CTC-positive pts by CellSearch had a statistically significant increased risk of distant recurrence (HR 4.93, p=0.0067). Moreover, pts who were CTC-positive by IE/FC had a statistically significant increased risk of breast cancer-specific death (HR=3.54, p=0.0138). DTC status, by itself, was not prognostic; however, when combined with CTC status by IE/FC (n=273), positive detection for both (CTC+DTC+) was significantly associated with increased risk of distant recurrence (HR=3.09, p=0.0270) and breast cancer-specific death (HR=4.55, p=0.0205). Table 1.Multivariate analysis to determine the prognostic significance of CTCs and DTCs detected at the time of surgery in treatment naive early breast cancer patients. Adjusted for age at diagnosis, tumor size, pathologic stage, HR and HER2 status, node status and grade.  DRFS  BCSS  Variable and Method% positiveHR [95% CI]Wald p-valueMedian f/u [range] Years*HR [95% CI]Wald p-valueMedian f/u [range] Years*CTC+ vs. CTC- by CellSearch94.93[1.56-15.6]0.00676.4 [0.16-13.8]4.50[0.76-26.5]0.09627.5 [0.71-15.0]CTC+ vs. CTC- by IE/FC401.92[0.93-3.95]0.07599.8 [0.09-18.5]3.54[1.29-9.72]0.013813.3 [1.93-18.5]DTC+ vs. DTC- by IE/FC181.46[0.75-2.81]0.26317.5 [0.09-18.5]1.48[0.64-3.42]0.35429.8 [1.55-18.5]CTC+DTC+ vs. CTC-DTC- by IE/FC8**3.09[1.14-8.40]0.02709.8 [0.09-18.5]4.55[1.26-16.39]0.020513.3 [1.93-18.5]*f/u - follow-up; **double positive Conclusions: We demonstrate the impact of quantitative evaluation of CTCs and DTCs by IE/FC. Our large single institution dataset, in which CTCs and DTCs have been contemporaneously quantitated, has the longest patient follow-up. Simultaneous detection of CTCs and DTCs at the time of definitive surgery in treatment naïve EBC pts is an independent prognostic factor associated with increased long-term risk of distant recurrence and death due to breast cancer. Given the lack of early endpoints for low-risk patients, liquid biopsy may be an important consideration for future studies. Citation Format: Magbanua MJM, Yau C, Wolf D, Lee JS, Chattopadhyay A, Scott JH, Yoder E, Hwang S, Alvarado M, Ewing CA, Delson AL, van't Veer L, Esserman L, Park JW. Detection of circulating tumor cells (CTC) in blood and disseminated tumor cells (DTC) in bone marrow at surgery identifies breast cancer patients (pts) with long-term risk of distant recurrence and breast cancer-specific death [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-02.