Response to neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) is highly prognostic and determines whether adjuvant chemotherapy is needed if residual tumor is found at surgery. To evaluate the predictive and prognostic values of circulating tumor DNA (ctDNA) in this setting, we analyzed tumor and serial bloods from 26 TNBC patients collected prior, during, and after NAC. Individual digital droplet PCR assays were developed for 121 variants (average 5/patient) identified from tumor sequencing, enabling ctDNA detection in 96% of patients at baseline. Mutant allele frequency at baseline was associated with clinical characteristics. Levels drastically fell after one cycle of NAC, especially in patients whose tumors would go on to have a pathological complete response (pCR), but then rose significantly before surgery in patients with significant residual tumor at surgery (p = 0.0001). The detection of ctDNA early during treatment and also late at the end of NAC before surgery was strongly predictive of residual tumor at surgery, but its absence was less predictive of pCR, especially when only TP53 variants are considered. ctDNA detection at the end of neoadjuvant chemotherapy indicated significantly worse relapse-free survival (HR = 0.29 (95% CI 0.08–0.98), p = 0.046), and overall survival (HR = 0.27 95% CI 0.075–0.96), p = 0.043). Hence, individualized multi-variant ctDNA testing during and after NAC prior to surgery has prognostic and predictive value in early TNBC patients.

Background: Circulating free DNA (cfDNA) is an exciting novel method to diagnose, monitor, and predict resistance and response to cancer therapies, with the potential to radically alter the management of cancer patients. To fulfill its potential, greater knowledge about preanalytical variables is required to optimize and standardize the collection process, and maximize the yield and utility of the small quantities of cfDNA extracted. Methods: To this end, we have compared the cfDNA extraction efficiency of three different protocols, including a protocol developed in house (Jewish General Hospital). We evaluated the impact on cfDNA levels of preanalytical variables including speed and timing of the second centrifugation and the use of k-EDTA and CTAD blood collection tubes. Finally, we analyzed the impact on fractional abundance of targeted pre-amplification and whole genome amplification on tumor and circulating tumor DNA (ctDNA) from patients with breast cancer. Results: Making use of a novel protocol for cfDNA extraction we increased cfDNA quantities, up to double that of commercial kits. We found that a second centrifugation at 3,000 g on frozen plasma is as efficient as a high-speed (16,000 g) centrifugation on fresh plasma and does not affect cfDNA levels. Conclusions: These results allow for the implementation of protocols more suitable to the clinical setting. Finally, we found that, unlike targeted gene amplification, whole genome amplification resulted in altered fractional abundance of selected ctDNA variants. Impact: Our study of the preanalytical variables affecting cfDNA recovery and testing will significantly enhance the quality and application of ctDNA testing in clinical oncology.

594 Background: TNBC, the most aggressive form of breast cancer, is treated primarily with chemotherapy, even before surgery (neoadjuvant chemotherapy or NAC). The prognosis and need for adjuvant therapy depends greatly on the tumor response assessed by pathology (pCR). Highly sensitive and specific ctDNA assays have been shown to be of prognostic value in the metastatic settingbut not yet in earlier settings. Methods: Tissue was collected from 26 Q-CROC-03 clinical trial TNBC patients before, during and after NAC, prior to surgery. Whole exome sequencing on tumor tissues was used to select single nucleotide variants with high allele frequency (VAF), prioritizing TP53, to generateindividual digital droplet PCR (ddPCR) assays. An average of 5 variants (range 1-12) per patient were tested, for a total of 121 variants. A detection threshold was defined for each variant from a pool of normal controls. Median follow-up was 55 months. Results: ctDNA was detectable in 96% of patients at baseline, but 20% of the 121 variants were not detectable at any time point. At baseline, the mean VAF of all analyzed variants, but not of TP53 variants alone, was significantly correlated (p < 0.05) with tumor factors (tumor size, stage, grade, nodal status before and at surgery, RCB score) but not with patient age or BRCA1/2 mutation status. 87 variants (74%) were detected at baseline and their VAF fell by 86% after 1 cycle of chemotherapy (T1). The detection of ctDNA at T1 was associated with DFS (p = 0.027) while the detection of ctDNA at the last post-chemotherapy pre-surgery time point (T4) was strongly associated with pathological complete response (pCR) and both DFS (p = 0.013) and OS(p = 0.006). At this time point, 5 of 41 variants (12%) were detected in pCR patients vs 42 of 80 (53%) in non-pCR, while only 6 of the 15 (40%) non-pCR patients had detectable TP53 variants. Interestingly, for variants detected at baseline, the positive predictive value of T4 ctDNA for disease recurrence was 69%, similar to that of non-pCR, while the negative predictive value of no ctDNA at T4 was 89% for disease recurrence vs 80% for pCR. Conclusions: ctDNA detection after NAC prior to surgery is strongly predictive of disease-free survival and overall survival and is comparable to pCR as a prognostic factor in our cohort (NCT01276899).

Background: Liquid biopsies to monitor response to treatment are a minimally invasive and highly attractive method for clinical application. Detection of ctDNA in plasma is now highly sensitive thanks to the use of novel highly sensitive and specific techniques such as ddPCR. In the present study we set out to analyze the utility of using ctDNA to monitor response to treatment in patients receiving standard neoadjuvant chemotherapy in triple negative breast cancer. Methods: Serial blood was collected from triple negative breast cancer patients participating in the Q-CROC-03 clinical trial (NCT01276899). The trial recruited triple negative breast cancer patients undergoing standard neoadjuvant chemotherapy. Paired biopsies were collected prior and at the end of treatment and serial bloods collected throughout the study. Whole exome sequencing was performed on tissues collected and we identified mutated genes of interest. Cell free DNA (cfDNA) was extracted from 3 ml of plasma and 4-10 variants per patient were analyzed by ddPCR in serial plasma samples collected before and during treatment. Response was measured by evaluating residual cancer burden (RCB), and non-responders were RCBII-III, responders RCB0-I. Results: For the present analysis, we identified 60 variants in tumors from 12 patients (9 RCBII-III and 3 RCB0-I). Except for TP53, none of the genes were shared among the tumors. 20% of the variants were not detected in ctDNA at any time point and we did not find any correlation between cfDNA levels and tumor size or response to treatment. The average variant allele frequency (VAF) of all detected variants at baseline was higher in RCBII-III patients than in RCB0-I patients (7.0 vs 0.7 respectively). Interestingly, variants that were detected either only in the pre-chemo tumor or in the post-chemo tumor were frequently detected throughout neoadjuvant therapy, highlighting the ability of ctDNA to capture tumor heterogeneity. In almost all cases, we observed a dramatic decrease in ctDNA VAF after one cycle of chemotherapy, including 30% to non-detectable levels. By the 5th cycle of chemotherapy 97% of detected variants had decreased (average 95% decrease). This decrease in ctDNA VAF was independent of RCB score. In some RCBII-III cases, ctDNA VAF increased prior to surgery, reflecting residual tumor presence. Conclusion: ctDNA could be detected in plasma of all early TNBC patients undergoing neoadjuvant chemotherapy with the majority of variants detected in plasma collected at baseline prior to chemotherapy. Once treatment started, the abundance of ctDNA markedly decreased in plasma independently of tumor response. The effect of chemotherapy on levels of ctDNA needs further investigation. Citation Format: Cavallone L, Adriana A-M, Aldamry M, Lafleur J, Cathy L, Alirezaie N, Bareke E, Majewski J, Ferrario C, Mihalciou C, Roy J-A, Markus E, Robidoux A, Pelmus M, Aleynikova O, Discepola F, Basik M. Dynamics of ctDNA changes during neoadjuvant chemotherapy in triple-negative breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-02.