Abstract 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.

Abstract Background: Neoadjuvant chemotherapy (NAC) is employed in patients with larger tumors to attempt to downstage locally advanced cancers to allow breast conservation and to assess in vivo tumor response. The Multi-Institutional Neoadjuvant Therapy MammaPrint Project I (MINT) study asked a secondary question of whether complete nodal downstaging could also be achieved with NAC. Methods: This analysis included 147 eligible invasive breast cancer patients with high tumor burdens, classified as cT2-4N0-3M0 (T2 greater than 3.5cm if N0). Patients who had a positive core biopsy and/or fine needle aspiration (FNA) on an axillary node prior to starting NAC were included in this analysis. Those who had a surgical sentinel lymph node biopsy were not included. Nodal involvement was established following neoadjuvant treatment by axillary lymph node dissection (ALND). Results: This population was 54% postmenopausal, average age 53 yrs (range 25 to 80 yrs). Tumor characteristics were 91% invasive ductal carcinoma; 65% T2, 29% T3, 6% T4; 87% LN1, 13% LN2-3; 3% low grade, 38% intermediate grade, 59% high grade; 65% ER-positive, 49% PR-positive, and 28% HER2-positive by immunohistochemistry; 84% High Risk (HR) and 16% Low Risk (LR) by MammaPrint (MP). After NAC, 45% (66/147) of these LN-positive patients were down-staged to ypN0 and also achieved a complete pathological response in the primary tumor. The potential for down-staging was inversely-related to tumor burden, where 47% (60/128) of N1, 35% (6/17) of N2, and 0% (0/2) of N3 patients were down-staged to ypN0. There were 3 patients who were down-staged (2 N2 to N1, and 1 N3 to N2), but not to ypN0. At surgery, 34% (44/128) of patients had no change, and 19% (24/129) progressed in LN staging. Pre vs Post NAC Nodal StagePre NAC Nodal StageypN0ypN1ypN2ypN3TotalcN16044222128cN2626317cN3 112Total6646296147 Conclusions: We confirmed that upon achieving a complete response of the primary tumor that there was also a pathologic complete response in the LN. About 53% of patients had no change or progression of LN involvement following NAC. Citation Format: Blumencranz P, Habibi M, Treece T, Blumencranz L, Yoder E, Audeh W, Carter E, McNaughton L, Roussos J, Shivers S, Acs G, Cox C, MINT Investigators Group. Neoadjuvant chemotherapy for breast cancer: Nodal downstaging is highly correlated with pathological complete response [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 PD8-04.

Abstract Background: IMPACt is a prospective, case-only study to measure the effect of MammaPrint (MP) and BluePrint (BP) on treatment decisions in breast cancer patients. Here, we report the results of the primary objective in women aged ≥18 years with histologically proven invasive stage I-II, hormone receptor (HR) positive, and HER2-negative breast cancer. Methods: The study included 369 women from 18 US institutions. The recommended treatment plan was captured before and after receiving results for MP and BP. Treatment was started after obtaining results. In addition to the effect of results on physician treatment decisions involving chemotherapy (CT) and physician confidence, the distribution of MP High Risk (HR) and Low Risk (LR) patients was also evaluated. Results: MP classified patients to 62% (n=228) LR and 38% (n=141) HR. Treatment decisions were changed for 25% (n=92) of women after receiving MP and BP results. Of the LR patients initially prescribed CT, 68% (45/66) had CT removed from their treatment recommendation. Of the HR patients who initially were not prescribed CT, 66% (42/64) had CT added. Overall, 89% (202/228) of LR patients did not receive CT, and likewise 84% (119/141) of HR patients did receive CT after receiving MP. Among those who did not change treatment (n=277), 68% of physicians reported having greater confidence in their prescribed therapy. Conclusions: The IMPACt trial shows MP generates a 25% overall treatment change in clinical practice. The highest impact is for women with LR results, where 68% are spared chemotherapy in favor of endocrine therapy alone. Additionally, 73% of physicians report having higher confidence in treatment decisions for their patient after MP. Table 1: Treatment changesTreatment Decision Pre- to Post-MPMP HRMP LRTotalCT to CT772198no CT tp CT42547CT to no CT04545no CT to no CT22157179Total141228369 Citation Format: Soliman H, Rehmus E, Shah V, Srkalovic G, Mahtani R, Levine E, Mavromatis B, Srinivasiah J, Kassar M, Gabordi R, Yoder E, Qamar R, Audeh W, IMPACt Investigators Group I. IMPACt trial: MammaPrint and BluePrint molecular subtyping guide treatment decisions in breast cancer [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 P6-13-04.

Abstract Background: Previous clinical trials have validated that the 70-gene signature MammaPrintTM provides prognostic and predictive information for early stage breast cancer patients and can identify low risk patients who may safely avoid adjuvant chemotherapy. Additionally, the neo-adjuvant I-SPY 1&2 TRIALs demonstrated that further stratification of patients into MammaPrint High 1 (MP1) and MammaPrint High 2 (MP2) risk groups may help predict chemo-sensitivity. There were significant differences in pathological complete response (pCR) rates for early stage, locally advanced breast cancer patients who were not HR+HER2- MammaPrint Low Risk. Specifically, the PARP inhibitor veliparib in combination with carboplatin recently graduated the I-SPY 2 phase 2 screening trial, having met the 85% predictive probability criterion with a triple-negative breast cancer signature, which was the subset recommended for this regimen's subsequent development. Given these data, we wanted to determine whether the Multi Institutional Neo Adjuvant Therapy MammaPrint Project (MINT) patient population confirmed the MP1/MP2 risk stratification, clarify if there is an associated receptor subtype for MP1/MP2 risk classes, and conclude if the stratification correlates to a significant difference in pCR. Methods: Array data from pre-treatment samples were obtained from 180 patients classified as MammaPrint High Risk, subtyped by IHC and treated with neo-adjuvant chemotherapy according to protocol. Response was measured by centrally assessed residual cancer burden pursuant to guidelines. Patients were then further stratified based on the MammaPrint Index per their classification threshold between MP1/MP2. Fisher's exact test was used to assess significance of association with pCR overall and within hormone receptor (HR) and HER2 subtypes. Results: MP1 vs MP2 risk classes yielded subsets with significant (p=0.007) differences in pCR. 44% (40/92) of MP2 patients achieved a pCR, compared to 24% (21/88) of MP1 patients. Next, we investigated whether the MP1 and MP2 risk classes were associated with receptor subtype. MP1 demonstrated a significant association and MP2 near significance. 32% (21/66) of triple-negative patients were classified as MP2 vs only 3% (2/66) MP1. Similarly, in the overall population, 28% (51/180) HR+HER2- are classified as MP1 vs 4% (8/180) MP2. Results in the pCR population were reflective of these subtype trends. 63% (58/92) of MP2 patients were classified triple-negative, of which nearly one quarter (21/92) had a measured pCR, whereas 58% (51/88) of MP1 patients were HR+HER2- with 3% (3/88) achieving pCR (Table1). Conclusion: This analysis in the MINT patient population supports previously published data and suggests that the MammaPrint High 1/2 risk classification may help predict chemo-sensitivity. Given the statistical significance of these data, we are currently investigating the biological mechanisms distinguishing the MP1/MP2 subgroups that may account for its use as a specific biomarker of response to chemotherapy treatment in future trials. Table 1.MP1MP2HER2+HER2-HER2+HER2-HR+HR-HR+HR-HR+HR-HR+HR-793241322185486456371514518818858Row#1= pCR, #2= RD, #3= Total Citation Format: Blumencranz LE, Shivers SC, Untch S, Treece TD, Yoder E, Blumencranz PW, Cox CE. MINT trial yields MammaPrint High1/High2 risk classes associated with significant differences in pCR and receptor subtype [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-16-05.

Background: Previous clinical trials have validated that the 70-gene signature MammaPrint TM provides prognostic and predictive information for early stage breast cancer patients and can identify low risk patients who may safely avoid adjuvant chemotherapy. Additionally, the neo-adjuvant I-SPY 1u00262 TRIALs demonstrated that further stratification of patients into MammaPrint High 1 (MP1) and MammaPrint High 2 (MP2) risk groups may help predict chemo-sensitivity. There were significant differences in pathological complete response (pCR) rates for early stage, locally advanced breast cancer patients who were not HR+HER2- MammaPrint Low Risk. Specifically, the PARP inhibitor veliparib in combination with carboplatin recently graduated the I-SPY 2 phase 2 screening trial, having met the 85% predictive probability criterion with a triple-negative breast cancer signature, which was the subset recommended for this regimen9s subsequent development. Given these data, we wanted to determine whether the Multi Institutional Neo Adjuvant Therapy MammaPrint Project (MINT) patient population confirmed the MP1/MP2 risk stratification, clarify if there is an associated receptor subtype for MP1/MP2 risk classes, and conclude if the stratification correlates to a significant difference in pCR. Methods: Array data from pre-treatment samples were obtained from 180 patients classified as MammaPrint High Risk, subtyped by IHC and treated with neo-adjuvant chemotherapy according to protocol. Response was measured by centrally assessed residual cancer burden pursuant to guidelines. Patients were then further stratified based on the MammaPrint Index per their classification threshold between MP1/MP2. Fisher9s exact test was used to assess significance of association with pCR overall and within hormone receptor (HR) and HER2 subtypes. Results: MP1 vs MP2 risk classes yielded subsets with significant (p=0.007) differences in pCR. 44% (40/92) of MP2 patients achieved a pCR, compared to 24% (21/88) of MP1 patients. Next, we investigated whether the MP1 and MP2 risk classes were associated with receptor subtype. MP1 demonstrated a significant association and MP2 near significance. 32% (21/66) of triple-negative patients were classified as MP2 vs only 3% (2/66) MP1. Similarly, in the overall population, 28% (51/180) HR+HER2- are classified as MP1 vs 4% (8/180) MP2. Results in the pCR population were reflective of these subtype trends. 63% (58/92) of MP2 patients were classified triple-negative, of which nearly one quarter (21/92) had a measured pCR, whereas 58% (51/88) of MP1 patients were HR+HER2- with 3% (3/88) achieving pCR (Table1). Conclusion: This analysis in the MINT patient population supports previously published data and suggests that the MammaPrint High 1/2 risk classification may help predict chemo-sensitivity. Given the statistical significance of these data, we are currently investigating the biological mechanisms distinguishing the MP1/MP2 subgroups that may account for its use as a specific biomarker of response to chemotherapy treatment in future trials. Citation Format: Blumencranz LE, Shivers SC, Untch S, Treece TD, Yoder E, Blumencranz PW, Cox CE. MINT trial yields MammaPrint High1/High2 risk classes associated with significant differences in pCR and receptor subtype [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-16-05.

Arachidonic acid metabolism is important in regulating the cerebral circulation and its response to CNS injury (1,2), a response that often involves vascular cell proliferation, inflammatory cell infiltrates, and glial cell reactions. Induction and regulation of cyclooxygenase-2 (COX-2) may be important in these processes. Since a number of pro-and anti-inflammatory cytokines, including TGFβ1, TNFα, and IL-1β, are produced during the course of CNS reaction to injury(3,4,5), we investigated the regulation of cerebrovascular COX-2 expression by these agents. In addition to cytokine production, the release of polyunsaturated fatty acids (PUFA), particularly arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3), is a common early reaction to CNS injury (6,7). Therefore, the potential interactions of PUFA and proinflammatory cytokines in the regulation of COX-2 were also investigated.

The purpose of this study was to determine whether the formation of docosahexaenoic acid in human cells occurs through a pathway that involves 24-carbon n-3 fatty acid intermediates and retroconversion. Normal human skin fibroblasts synthesized radiolabeled docosahexaenoic acid from [1-(14)C]18:3n-3, [3-(14)C]22:5n-3, [3-(14)C]24:5n-3, and [3-(14)C]24:6n-3. The amount of docosahexaenoate formed was reduced in fibroblasts defective in peroxisomal biogenesis, by 90-100% in Zellweger's syndrome and by 50-75% in infantile Refsum's disease. Fatty acid elongation and desaturation were intact in these mutant cells. No decrease in radiolabeled docosahexaenoic acid production occurred in mutant fibroblasts defective in peroxisomal alpha-oxidation or mitochondrial beta-oxidation, or in normal fibroblasts treated with methyl palmoxirate to inhibit mitochondrial beta-oxidation. Therefore, the retroconversion step in docosahexaenoic acid formation occurs through peroxisomal beta-oxidation in normal human cells. These results demonstrate that the pathway for docosahexaenoic acid synthesis in human cells involves 24-carbon intermediates. The limited ability to synthesize docosahexaenoic acid may underlie some of the pathology that occurs in genetic diseases involving peroxisomal beta-oxidation.

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood-brain barrier in this process, cultured murine cerebromicrovascular endothelia were incubated with [1-14C]18:2 omega-6 or [1-14C]18:3 omega-3 and their elongation/desaturation products determined. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary product from 18:3 omega-3 was 20:5 omega-3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4 omega-6 and 20:5 omega-3 that were formed. No 22:5 omega-6 or 22:6 omega-3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3 omega-3 and 18:2 omega-6 was nearly identical, 18:3 omega-3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3 omega-3 by the elongation/desaturation pathway. These findings suggest that the blood-brain barrier can play an important role in the elongation and desaturation of omega-3 and omega-6 essential fatty acids during their transfer from the circulation into the brain.