High-risk Triple-negative Breast Cancer Research Articles

Abstract P2-14-07: Association between initiation of adjuvant chemotherapy beyond 30 days following surgery and overall survival among patients with triple-negative breast cancer

Abstract Background: The optimal time between surgery and initiation of adjuvant chemotherapy is unknown, though delayed time to chemotherapy (TTC) is associated with decreased outcomes of breast cancer patients. Recently, studies have suggested that the association might be subtype-dependent and that TTC within 30 days should be warranted particularly in high-risk triple-negative breast cancer (TNBC) patients. Objective: To determine the extent to which TTC beyond 30 days is associated with reduced overall survival (OS) in TNBC patients. Methods: Using the population-based nationwide Netherlands Cancer Registry we identified TNBC patients diagnosed between 2006 and 2014 who received adjuvant chemotherapy. We distinguished between patients who underwent breast-conserving surgery (BCS) versus mastectomy given the difference in preoperative characteristics and outcomes. Median (95% confidence interval) follow-up was 82.9 (80.5-86.5) and 81.4 (79.5-83.9) months, respectively. Main outcomes and measures: The association between TTC beyond 30 days and OS was estimated with hazard ratios (HR) using propensity-score matched Cox proportional hazard analyses separately for patients who underwent BCS and mastectomy. Results: In total, 3016 patients were included, of whom 1079 (35.8%) underwent BCS and 1937 (64.2%) underwent a mastectomy. In matched patients who underwent BCS, 10-year OS was significantly better for patients with TTC within 30 days compared to patients with TTC beyond 30 days (84.4% vs. 76.9%, P=0.001). Patients with TTC beyond 30 days were more likely than those with TTC within 30 days to die within 10 years after surgery (HR 1.69 (95% CI 1.22-2.34), P=0.002). In matched patients who underwent mastectomy, there was no difference in 10-year OS between those with TTC within or beyond 30 days (74.5% vs. 74.7%, P=0.716), nor an increased risk of death for those with TTC beyond 30 days (HR 1.04 (95%-CI 0.84-1.28), P=0.716). In both populations, the associations were independent of adjuvant radiotherapy. Conclusions: The current results suggest that initiation of chemotherapy beyond 30 days is associated with decreased OS in TNBC patients who underwent BCS; no association was observed for patients who underwent a mastectomy. Therefore, timelier initiation of chemotherapy in TNBC patients undergoing BCS seems warranted. Citation Format: Erik Heeg, Perla J. Marang-van de Mheen, Marissa C. Van Maaren, Kay Schreuder, Rob A.E.M. Tollenaar, Sabine Siesling, Monique E.M.M. Bos, Marie-Jeanne T.F.D. Vrancken Peeters. Association between initiation of adjuvant chemotherapy beyond 30 days following surgery and overall survival among patients with triple-negative breast cancer [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 P2-14-07.

Dose-dense paclitaxel plus carboplatin vs. epirubicin and cyclophosphamide with paclitaxel as adjuvant chemotherapy for high-risk triple-negative breast cancer.

ObjectiveThe objective of this open-label, randomized study was to compare dose-dense paclitaxel plus carboplatin (PCdd) with dose-dense epirubicin and cyclophosphamide followed by paclitaxel (ECdd-P) as an adjuvant chemotherapy for early triple-negative breast cancer (TNBC).MethodsWe included Chinese patients with high recurrence risk TNBC who underwent primary breast cancer surgery. They were randomly assigned to receive PCdd [paclitaxel 150 mg/m2 on d 1 and carboplatin, the area under the curve, (AUC)=3 on d 2] or ECdd-P (epirubicin 80 mg/m2 divided in 2 d and cyclophosphamide 600 mg/m2 on d 1 for 4 cycles followed by paclitaxel 175 mg/m2 on d 1 for 4 cycles) every 2 weeks with granulocyte colony-stimulating factor (G-CSF) support. The primary endpoint was 3-year disease-free survival (DFS); the secondary endpoints were overall survival (OS) and safety. ResultsThe intent-to-treat population included 143 patients (70 in the PCdd arm and 73 in the ECdd-P arm). Compared with the ECdd-P arm, the PCdd arm had significantly higher 3-year DFS [93.9% vs. 79.1%; hazard ratio (HR)=0.310; 95% confidence interval (95% CI), 0.137−0.704; log-rank, P=0.005] and OS (98.5% vs. 92.9%; HR=0.142; 95% CI, 0.060−0.825; log-rank, P=0.028). Worse neutropenia (grade 3/4) was found in the ECdd-P than the PCdd arm (47.9% vs. 21.4%, P=0.001). ConclusionsPCdd was superior to ECdd-P as an adjuvant chemotherapy for early TNBC with respect to improving the 3-year DFS and OS. PCdd also yielded lower hematological toxicity. Thus, PCdd might be a preferred regimen for early TNBC patients with a high recurrence risk.

Dose-Dense Paclitaxel Plus Carboplatin (PCdd) versus Epirubicin and Cyclophosphamide (ECdd) Followed by Paclitaxel (P) As Adjuvant Chemotherapy in Early Triple-Negative Breast Cancer at High Risk of Recurrence, A Randomised, Open-Label, Phase 3 Trial

Background: Triple-negative breast cancer (TNBC) may be more sensitive to platinum-based regimens. This open-label, randomized phase III trial aimed to compare dose dense paclitaxel plus carboplatin (PCdd) with dose-dense epirubicin and cyclophosphamide followed by paclitaxel (ECdd-P) regimen as adjuvant chemotherapy (AC) for high-risk early TNBC. Methods: We included Chinese patients with high recurrence risk TNBC who underwent primary breast surgery. They were randomly assigned in a 1:1 ratio to receive PCdd (paclitaxel 150 mg/m2 on day 1 and carboplatin AUC=3 on day 2 for 8 cycles) or ECdd-P (epirubicin 80 mg/m2 divided in 2 days and cyclophosphamide 600 mg/m2 on day 1 for 4 cycles followed by paclitaxel 175 mg/m2 on day 1 for 4 cycles) every 2 weeks, with granulocyte colony-stimulating factor (G-CSF) support. The primary endpoint was 3-year disease-free survival (DFS); the secondary endpoints were overall survival (OS) and safety. Findings: The intent-to-treat population included 143 patients (70 in the PCdd arm and 73 in the ECdd-P arm). Compared with the ECdd-P arm, the PCdd arm had significantly higher 3-year DFS (93⋅9% vs.79⋅1%, hazard ratio[HR]=3⋅224, 95% confidence interval [CI] =1⋅420-7⋅321, log-rank p=0⋅0051) and OS (98⋅5% vs.92⋅9% [HR] =7⋅092, 95% CI=1⋅212-16⋅630, log-rank p=0⋅0281). The 3-year DFS in the PCdd arm was significantly higher (p 40 years, Ki-67>30, and clinically evaluated lymph nodes. Both regimens were well tolerated. Worse neutropenia (grade 3/4) was found in the ECdd-P arm (47⋅9% vs. 21⋅4%, p=0⋅001). Interpretation: PCdd was superior to ECdd-P as AC for early TNBC with respect to improving the 3-year DFS and OS. PCdd also yields lower hematological toxicity.Thus, PCdd might be a preferred AC regimen for early TNBC patients with high recurrence risk. Trial Registration: This trial is registered at ClinicalTrials. gov (number NCT01378533). Funding Statement: This study was supported by National Key Research and Development Program of China (2018YFC1312101); and Chinese Academy of Medical Science Initiative funded this study for Innovative Medicine (CAMS-12M-1-010). Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: All interventions were performed in accordance with Chinese laws and regulations and the Helsinki declaration. The study protocol was approved by the independent ethics board committee at the National Cancer Center, Chinese Academy of Medical Sciences (Approval number: CH-BC-012).

208P - Real-world comparison of the impact of adjuvant capecitabine in women with high-risk triple-negative breast cancer after neoadjuvant chemotherapy

BackgroundAdjuvant capecitabine (adjC) is recommended for patients with triple-negative breast cancer (TNBC) and poor response to neoadjuvant chemotherapy. Despite evidence of benefit, this therapy is not available in situations of limited resources, such as in the Brazilian public health system. MethodsA retrospective observational cohort study was conducted in Princess Margaret (PM) Cancer Centre in Toronto, Canada, and in A.C.Camargo Cancer Center (AC) in São Paulo, Brazil. Patients with TNBC who had completed neoadjuvant chemotherapy (taxane and/or anthracycline) followed by surgery, with a high residual cancer burden (RCB-II or RCB-III) and received at least 1 cycle of adjC in PM or had active surveillance (AS) in AC were evaluated. A descriptive comparison was made between PM and AC. Recurrence free survival (RFS), overall survival (OS), and safety profile were explored descriptively. Chi-Square test and Mann-Whitney test were used to assess categorical and continuous variables data, while Kaplan-Meier survival and Cox-regression were used for time-to-event analyses. ResultsFrom 06/17 to 03/19, 21 patients received adjC in PM and from 04/02 to 03/19, 70 patients had AS in AC. Starting dose of capecitabine was 1000mg/m² bid. Seven patients (43.8%) completed 8 cycles, two (12.5%) received 6 cycles and for the remaining patients therapy is ongoing. The median follow-up was 23 months (range 1.9-36 months). At PM 76.2% of the patients were free from recurrence or death and 81% were alive at 3 years. The mean RFS was better in the adjC group (mean 30.8 months vs 20.0 months HR: 0.23; p=0.045). Mean OS was 29 months in adjC group vs 28 months in AS (HR: 1.48; p=0.610). Common adverse events were hand-foot syndrome, fatigue, diarrhea, nausea and anemia. Dose reduction, delay to next chemotherapy cycle or discontinuance of treatment were necessary in 13 (61.9%) patients. ConclusionsOutcome of patients receiving adjC at PM was better than those on AS in AC, suggesting benefit from this treatment in a real-world setting. AdjC is an inexpensive, widely available and well tolerated oral chemotherapy and should be considered an option for improvement of care even in public health systems of less developed countries. Legal entity responsible for the studyThe authors. FundingThis work has been supported by a UICC Technical Fellowship. DisclosureE. Amir: Speaker Bureau / Expert testimony: Genentech/Roche; Advisory / Consultancy: Apobiologix; Advisory / Consultancy: Agendia; Advisory / Consultancy: Myriad Genetics. All other authors have declared no conflicts of interest.

Precision Medicine in Triple Negative Breast Cancer: The Future is Coming

Abstract Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer, with high recurrence rate and short survival after metastasis. However, TNBC is a pathologically and clinically heterogenous disease. Chemotherapy is currently the main treatment choice for TNBC. Tremendous effort has been devoted to identifying new treatment regimen in high-risk or metastatic TNBC patients. Recent studies have classified TNBC into several distinct molecular subtypes and proposed new treatment regimen. In this presentation, the strategies to identify high-risk and decrease relapse in early TNBC patients will be discussed. Furthermore, new treatment strategies in specific subtypes of TNBC patients will be presented, including anti-androgen in androgen receptor-positive TNBC patients, PARP inhibitors in BRCA mutant or High HRD patients, combination immunotherapy, and other approaches. There has never been a better time for precise treatment in TNBC patients.

Loss of PICH promotes chromosome instability and cell death in triple-negative breast cancer

Triple-negative breast cancer (TNBC), defined by the lack of expression of estrogen, progesterone, and ERBB2 receptors, has the worst prognosis of all breast cancers. It is difficult to treat owing to a lack of effective molecular targets. Here, we report that the growth of TNBC cells is exceptionally dependent on PICH, a DNA-dependent ATPase. Clinical samples analysis showed that PICH is highly expressed in TNBC compared to other breast cancer subtypes. Importantly, its high expression correlates with higher risk of distal metastasis and worse clinical outcomes. Further analysis revealed that PICH depletion selectively impairs the proliferation of TNBC cells, but not that of luminal breast cancer cells, in vitro and in vivo. In addition, knockdown of PICH in TNBC cells induces the formation of chromatin bridges and lagging chromosomes in anaphase, frequently resulting in micronucleation or binucleation, finally leading to mitotic catastrophe and apoptosis. Collectively, our findings show the dependency of TNBC cells on PICH for faithful chromosome segregation and the clinical potential of PICH inhibition to improve treatment of patients with high-risk TNBC.

Abstract P5-12-04: A new method of data analysis to derive DNA methylation signatures that stratify risk of recurrence in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) accounts for 10-17% of all breast cancer and is more likely to be of higher histological grade, poorly differentiated, associated with a higher recurrence rate and with decreased overall survival. The clinical course of a TNBC patient remains difficult to predict, as tumors with homogenous morphological characteristics may vary in response to therapy and have divergent outcomes. Therefore, additional analytical methods are needed to better classify TNBC. Our goal is to refine the analysis of methylome datasets to derive reliable molecular signatures that can distinguish TNBC patients with good outcomes who may benefit from less aggressive treatment, from those with poor outcomes who would be candidates for more aggressive treatments. Methods: Our laboratory has conducted and reported, in this meeting, results from analysis of 450k methylation array data on a discovery set of 53 high-risk TNBC cases and 62 low-risk controls treated by locoregional therapy alone, as well as 5 normal breast tissue samples. High-risk cases were defined as patients that relapsed within 0.5 to 6.5 years from the time of diagnosis, while low-risk controls had no relapse and >4 year recurrence-free intervals (RFI). In this work, we devised and applied a novel methylation biomarker discovery program named Hypermethylated Outlier Detector (HOD) that emphasizes the selection of highly methylated markers in cases compared to controls, to find a high-risk signature in the TNBC discovery set. The methylation signature identified by HOD was interrogated in a test set of 50 TNBCs (with 16 recurrences) that did not receive chemotherapy, and in a second test set of 131 TNBCs (with 33 recurrences) that did receive chemotherapy. Results: HOD identified 39 hypermethylated markers (beta >0.20) that could accurately distinguish between the high-risk cases and the low-risk controls in the discovery set of TNBCs (n=115) treated with locoregional therapy alone. In the test set of TNBC (n=50) with no chemotherapy the 39 markers distinguished high from low risk individuals (likelihood ratio test P=0.049). In a second test set of TNBC (n=131) that received chemotherapy the 39 hypermethylated markers again distinguished high from low risk individuals (likelihood ratio test P=0.0043). Conclusions: We have presented evidence that a methylation signature identified by HOD can be used to identify TNBC patients that have a high-risk of relapse regardless of receiving chemotherapy. This methylation signature could potentially be used to inform physician decisions on therapeutic strategies for TNBC patients. This could ultimately lead to less aggressive treatment given to patients possessing a methylation profile consistent with a better prognosis. Conversely, patients with hypermethylation in the 39 markers will likely benefit from a more aggressive course of treatment. Citation Format: Downs BM, Cope LM, Fackler MJ, Cho S, Wolff AC, Regan MM, Sukumar S, Umbricht CB. A new method of data analysis to derive DNA methylation signatures that stratify risk of recurrence in triple negative 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 P5-12-04.

CXCL13 inhibition induce the apoptosis of MDA-MB-231 breast cancer cells through blocking CXCR5/ERK signaling pathway.

Treatment of the high-risk triple negative breast cancer (TNBC) is a critical clinical challenge. Here we aimed to explore a novel strategy for TNBC treatment by blocking the tumor-associated chemokine CXCL13 in the MDA-MB-231 TNBC cells. MDA-MB-231 cells were treated with anti-CXCL13 antibodies (inhibition group), or phosphate-buffered saline (PBS) (control group), followed by determining the levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta-1 (TGF-β1) with enzyme-linked immunosorbent assay (ELISA). The effects of CXCL13 inhibition on cell proliferation and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Quantitative Real Time-PCR (qRT-PCR) and Western blot were used to compare the levels of CXCL13, CXCR5, extracellular signal-regulated kinase (ERK). The levels of cyclin D1 and cleaved caspase-9 were detected by Western blot. The levels of IL-1, TNF-α and TGF-β1 in MDA-MB-231 cells treated with anti-CXCL13 antibodies were significantly downregulated (p<0.05). Meanwhile, CXCL13 blockade decreased the cell proliferation and increased the apoptosis rate of MDA-MB-231 cells. The inhibition of CXCL13 led to marked reduction in CXCL13 and CXCR5 mRNA and an increase in ERK mRNA. The inhibition of CXCL13 resulted in the downregulation of CXCL13, CXCR5, p-ERK/ERK, cyclin D1 and upregulation of cleaved caspase-9 proteins. CXCL13 blockade effectively suppressed the proliferation of MDA-MB-231 cells by promoting cell apoptosis. This effect is presumably associated with the downregulation of CXCL13 and suppression of the CXCR5/ERK signaling pathway.

CHST9 rs1436904 genetic variant contributes to prognosis of triple-negative breast cancer

Triple-negative breast cancer (TNBC) refers to one aggressive histological subtype of breast cancer with high heterogeneity and poor prognosis after standard therapy. Lack of clearly established molecular mechanism driving TNBC progression makes personalized therapy more difficult. Thus, identification of genetic variants associated with TNBC prognosis will show clinic significance for individualized treatments. Our study is aimed to evaluate the prognostic value of the genome wide association study (GWAS)-identified CHST9 rs1436904 and AQP4 rs527616 genetic variants in our established early-stage TNBC sample database. Cox regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). CHST9 rs1436904G allele was significantly associated with decreased disease-free survival time (DFS) (8.5 months shorter in GG genotype carriers compared to TT genotype carriers, HR = 1.70, 95% CI = 1.03–2.81, P = 0.038). Stratified analyses showed an increased risk of cancer progression in CHST9 rs1436904G allele carriers harboring larger tumor (tumor size > 2 cm), without lymph-node metastasis, being premenopausal at diagnosis or with vascular invasion (P = 0.032, 0.017, 0.008 or 0.003). Our findings demonstrate that the GWAS-identified 18q11.2 CHST9 rs1436904 polymorphism significantly contributes to prognosis of early-stage TNBC, suggesting its clinical potential in the screening of high-risk TNBC patients for recurrence and the possibility of patient-tailored therapeutic decisions.

A functional BRCA1 coding sequence genetic variant contributes to prognosis of triple-negative breast cancer, especially after radiotherapy.

As a subtype of breast cancer, triple-negative breast cancer (TNBC) shows poorprognosis and high heterogeneity. Precise identification of TNBC subgroups relevant to clinical prognosis is crucial in the design and administration of individualized treatments.This study aimed to evaluatethe prognostic value of the functionalBRCA1rs799917genetic variant in TNBC. Associations between the rs799917 polymorphism and progression risk were investigated after genotyping 370 TNBC patients. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated by Cox regression. RESULTS: We found that the rs799917T allele was associated with a significantly increased risk of diseaseprogressionand shortened progression-free survival time (PFS) (P = 0.001 for log-rank test). Notably,TNBC patients with thers799917 CC genotype showed about 22 months prolonged PFS compared to the TT genotype after radiotherapy (HR 4.44, 95% CI 1.98-9.93;P = 2.9×10-4). Additionally, in overweight patients, the mean PFS of the rs799917TT genotype was 10 months shorter than that of the CC genotype (HR 3.57, 95% CI 1.46-8.73,P = 0.005). Our findings demonstrate that the functionalBRCA1genetic variant contributes to prognosis of TNBC. Our study also highlights the clinical potential of this polymorphism in the screening of high-risk TNBC patients for recurrence and the possibility of patient-tailored decisions especiallyduringradiotherapy.

Long term follow up of patients with locally advanced triple negative breast cancer treated with adjuvant high dose chemotherapy and autologous stem cell transplantation: A single center experience.

e12590 Background: Adjuvant high dose chemotherapy (HDC) with Autologous Hematopoietic Stem Cell Transplantation (AuSCT) as part treatment of high risk locally advanced breast cancer has remained controversial. Multiple trials reported disease free survival (DFS) without Overall survival (OS) resulting in its abandonment in early 2000s. However, post hoc analysis of these trials consistently reported DFS and OS benefit in young and triple negative breast cancer (TNBC) subgroups. This has not been re-evaluated till date. Recent European registry reports coupled with improved transplant related mortality (TRM) and still poor out-come of standard of care in TNBC subgroup has generated renewed interest. We report long term out-come of locally advanced high risk TNBC treated with HDC and AuSCT treated in Karmanos Cancer Institute from 1995 to 2001 Methods: Majority of the patients were treated with Adriamycin and Taxane based induction chemotherapy. Patients without evidence of metastatic disease proceeded to HDC and AuSCT using Carmustine 600mg/sqm Cyclophosphamide 5.6gm/sqm and Cisplatin 165mg/sqm (STAMP 1 regimen). This is followed with loco- regional radiation per protocol. Results: 72 hormone negative patients with Lymph Node(LN) &gt; 4 or inflammatory breast cancers were selected from 576 treated for advanced or metastatic breast cancer. 33 patients were TNB with HER2 status of 39 patients unknown. Median time from diagnosis to stem cell transplantation was 6 months. Median age at diagnosis was 47yrs. Mean LN involvement was 9 with 90% having (4-20) LN positivity. With median follow up of 16 years,10yrs DFS and OS were both 62.5%. Median follow up for DFS and OS was not reached.TRM was 9% mostly from pulmonary toxicity. Conclusions: This study of locally advanced high risk TNBC treated with adjuvant HDC and AuSCT have high 10yr OS of 62.5% compared to current standard of care. With the current improvement in TRM, reevaluation of this strategy through clinical trials in this subgroup whose outcome remain poor is reasonable.

Association of family history and location of BRCA1 and BRCA2 mutations in triple-negative breast cancer patients.

e12588 Background: We assessed the prevalence of family history and its association with germline BRCA1/2mutation status/location and age at onset in triple-negative breast cancer (TNBC) patients. Methods: 266 patients with TNBC &lt; 60 years unselected for family history of cancer were enrolled and germline DNA was sequenced to identify mutations. Family pedigrees were prospectively collected from these patients. Logistic regression was used to investigate family history and its association with mutation type/location and age at onset. ROC curves were constructed to determine good predictors of BRCAmutations. Results: BRCA mutations were identified in 18.0% of all patients (15.0% BRCA1, 3.0% BRCA2). BRCA1 carriers have a significantly earlier age at onset than non-mutation carriers (40 vs 49 years; p &lt; .001). While 39/124 (31.4%) patients with family history of cancer carried a BRCA1/2 mutation, 9/142 (6.3%) BRCA carriers had no family history of cancer. BRCA1 carriers with ≥1BC in the family are commonly identified in the breast cancer cluster regions (53.1%). BRCA2 carriers more commonly cluster within the ovarian cancer regions. Of note, this difference was not statistically significant. Women with mutations in BRCA1 OCCR are diagnosed at a younger age. TNBC diagnosed ≤45 years with ≥1BC and ≥1OC in the family are good predictors of BRCA1 mutation (AUC 0.867). Conclusions: Young women with TNBC and a family history of BC and OC are likely to have a BRCA mutation. Specific BRCA mutation locations may add to the identification of a subgroup of TNBC patients with a relatively higher risk of subsequent ovarian cancer. Identification of high-risk TNBC patients with BRCA1 mutation will enable clinicians to optimize cancer management for this phenotype, but will require further validation in larger studies.

Abstract B08: Identifying high-risk triple negative breast cancer patients using a novel cycling kinetics metric

Abstract Background Ki67 Index (KI) and Mitotic Index (MI) are proliferation markers with established prognostic value in breast cancer. These indices are evaluated individually and on disparate measurement scales; they thereforefail to capture information about cell cycling kinetics of proliferating cells. Within the triple negative (TNBC) subtype , we rationally integrate the two markers to identify high-risk patients whose proliferative cells exhibit fast cycling kinetics. Methods Pathology reports of breast cancer patients (n=10,504 from Northside Hospital, Atlanta and n=1560 from Nottingham Hospital, UK) were retrospectively analyzed for mitotic scores, KI and clinical outcomes. Mitotic counts in 267 H&amp;E-stained breast carcinoma samples were evaluated by two pathologists to transform mitotic scores into %MI based on cellularity. %MI: %KI ratio was defined as the Ki67-Adjusted Mitotic Score (KAMS), which reflects cycling kinetics of proliferative cells. Ability of KAMS to stratify triplenegative breast cancers (TNBCs) was tested in three cohorts who received only adjuvant chemotherapy (n=478 from Northside Hospital, USA; n=322 from Nottingham Hospital, UK, and n=108 from OlabisiOnabanjo University, Nigeria). Stratification of KAMS, KI and MI were performed onthe thresholdsthat produced the lowest AIC (best model fit).Slow-cycling and fast-cycling TNBC subgroups from Nottingham Hospital were analyzed for biomarker expression. Results Kaplan-Meier survival analyses, AIC andc2 values showed that KAMS-based stratification of TNBCs into two subgroups was superior to that by either KI or MI, regardless of hospital, and KAMS retained its significance in multivariate analyses, controlling for stage and age. Fast-cycling TNBCs have poorer prognosis than slow-cycling TNBCs, perhaps due to higher intratumoral heterogeneity in fast cycling tumors. Fast-cycling TNBCs showed high expression of proteins implicated in DNA damage response, sumoylation, EGFR signaling and metastasis. By contrast, slow-cycling TNBCs showed extensive chromatin modification. Conclusion KAMS quantifies cell cycling kinetics, stratifies TNBCs and yields new risk-predictive information that is not revealed by either KI or MI. KAMS reveals the underlying heterogeneity in cycling kinetics among TNBCs and helps identify TNBCs who might benefit from treatments that target the cell cycle machinery. Citation Format: Sergey Klimov, Guanhao Wei, Andrew Green, Mohammed Aleskandarany, Emad Rakha, Ian Ellis, Guilherme Cantuaria, Ayodeji O. Agboola, Michelle Reid, Li Xiaoxian, Rida C. G. Padmashree, Remus Osan, Ritu Aneja. Identifying high-risk triple negative breast cancer patients using a novel cycling kinetics metric. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B08.

Abstract P3-07-34: Breast cancer pathologic subtype and fetal microchimerism

Abstract Background: Stably persistent offspring-origin cells in a woman’s circulation and tissues decades after pregnancy, also known as fetal microchimerism, is deficient in women with biologically unselected breast cancer and suggests a protective role. Although the finding is in harmony with the well-known association of parity with protection against future breast cancer, it has recently emerged that certain tumor types such as triple negative breast cancer are positively associated with parity. Here we tested the hypothesis that fetal microchimerism is positively associated with triple negative breast cancer in young women. Methods: Buffy coat specimens were obtained from a subset (n=450) of pathologically confirmed low-risk luminal and high-risk triple negative breast cancer patients and control participants from a population-based cohort study of all women aged 20-44 diagnosed with invasive breast cancer in the three county Seattle-Puget Sound metropolitan area from 2004-2010. Using quantitative PCR, DNA extracts from these specimens were tested for the presence and concentration of Y chromosome sequence DYS14, a marker of male fetal microchimerism. Results: At this interim analysis of 405 specimens, 29.3% (56/191) of the healthy controls tested positive. Using controls as a reference group, 37.8% (34/90; OR .68; 95% CI .39-1.2) of women who developed luminal breast cancer tested positive for the presence of DYS14, while 26.6% (33/124; OR 1.14; 95% CI .67-1.96) of women who developed triple negative breast cancer tested positive. Discussion: A preliminary analysis does not suggest an association between fetal microchimerism and breast cancer pathologic subtype in young women. Additional analyses are pending and will be presented at the meeting. Young women may regulate fetal microchimerism differently and surprisingly our data may suggest that as fetal microchimerism emerges later in life in the circulation, so does the protection against breast cancer – a finding consistent with population based studies of parity as a risk factor for breast cancer. Citation Format: David A Mahoney, Veronica L Winget, VK Gadi, Christopher I Li, Peggy L Porter, Jean A McDougall. Breast cancer pathologic subtype and fetal microchimerism [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-07-34.

Abstract PR13: Using a hypoxia-induced CSC tumor initiation model to improve predictive potential for patients with metastatic breast cancer

Abstract About 10-20% of breast cancers are found to be triple-negative. TNBCs have a relapse pattern that is very different from hormone-positive breast cancers: the risk of relapse is much higher for the first 3-5 years but drops substantially below that of hormone-positive breast cancers after that. Therefore, preventing tumor recurrence and metastasis in high-risk TNBC patients within 3-5 years of diagnosis will dramatically improve the long-term survival of these patients. Currently, most of preclinical animal models have limited predictive clinical potential for advanced metastatic diseases. One possible explanation is that most of the pre-clinical studies focus on the therapeutic benefit of eradicating bulk tumor cells. Using a stem-like subpopulation from triple negative breast cancer (TNBC) cell lines enriched by exposing the cells to cycles of hypoxia and reoxygenation, we showed that mammary tumors initiated from this subpopulation have a high recurrence rate (100%) after the taxol treatment compared to no recurrence from taxol treated tumors initiated from the bulk cancer cells. Our model mimics the progression of recurrence-prone breast tumors with a period of dormancy before the recurrence of local and distal tumors. Molecular analysis of the treatment naïve and recurrent tumors revealed increased expression of chemoresistance-associated miRNAs in the recurred xenograft tumors. In addition, we identified new markers in the recurred xenograft tumors that are also increased in drug resistant human breast tumors compared to the treatment naïve breast tumors. Ultimately, we propose that cancer stem cell based tumor model can be used to improve the predictive potential of pre-clinical models for patients with metastatic breast tumors. This abstract is also presented as Poster A12. Citation Format: Purvi Patel, Yupo Ma, Haiyan Zhai, Jingfeng Ju, Emily Chen. Using a hypoxia-induced CSC tumor initiation model to improve predictive potential for patients with metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR13.

Abstract P3-05-15: TP53 mutation and nestin protein expression patterns affect the outcome of patients with triple negative breast cancer (TNBC)

Abstract Background-aim: Nestin has recently been proposed as an ideal positive biomarker for calling basal-like breast cancer (BL). BL and TNBC overlap in 70-80% of cases and, although very heterogeneous at the genomic and phenotypic levels, most of these carry TP53 mutations. Herein, we investigated the effect of TP53 mutations and Nestin expression on the outcome of early high-risk TNBC patients who had received adjuvant chemotherapy (anthracyclines and/or taxanes). Methods: TP53 mutations were assessed in 190 FFPE TNBC DNA samples with massive parallel sequencing (entire coding region) in Ion Torrent PI chips. Variant calling and annotation (Ion Reporter 1.6) was followed by stringent read quality filtering (p&amp;lt;0.001). Deleterious and hot-spot mutations (Ingenuity &amp; Oncomine databases) in 183 informative tumors were accepted for analysis. Nestin protein expression was examined by immunohistochemistry (IHC) in 283 centrally assessed TNBC on tissue microarrays; positivity was set at 1% of cells regardless of staining intensity. Further BL and TNBC related IHC markers were also examined. Results: Nestin was expressed in 134/283 tumors (47.3%), more frequently in BL-TNBC (CK5+ and/or EGFR+, Fisher's exact p&amp;lt;0.001) and in highly proliferative tumors (p&amp;lt;0.001), while it was positively associated with high tumor grade (p = 0.017) and with P-cadherin IHC (p = 0.001). TP53 mutations were observed in 141/183 tumors (77%), out of which 30 carried truncating TP53 mutations (21.3%) and 59 double damaging mutations (41.8%) usually at similar coverage; among these, truncating mutations occurred in 16 cases. TP53 mutations were more common in BL-TNBC (p = 0.029). Double mutations were positively associated with E- and P-cadherin protein expression (p = 0.016 and p = 0.020, respectively) Although TP53 mutations in general and the presence of double mutations were not associated with p53 IHC, tumors with truncating mutations were almost exclusively p53 IHC negative (p&amp;lt;0.001) in line with the detection capacity of the employed antibody. As single markers, neither Nestin nor TP53 mutations (any pattern) had significant effects on patient outcome. When combined, a DFS-related interaction between Nestin and double TP53 mutations was observed (Wald's p = 0.016). Nestin positive tumors with double TP53 mutations (n = 22) conferred median DFS of 45 mo. This interval was significantly shorter as compared to that of patients with Nestin positive tumors without double TP53 mutations (n = 62; median DFS 56 mo; HR 2.2; 95%CI 1-4.5; p = 0.045) and Nestin negative tumors with double TP53 mutations (n = 25) that were associated with the most favorable DFS (median: 66 mo; HR 4; 95%CI 1.3-12.4; p = 0.016). Nestin negative tumors without double TP53 mutations performed similarly to both positive tumors, although not statistically significant. Conclusions: The presence of double TP53 mutations in TNBC may reflect the genetic instability and heterogeneity characterizing these tumors. This feature in combination with the putative cancer stem cell and BL marker Nestin seems to aggravate the outcome of TNBC patients treated with standard adjuvant chemotherapy regimens. These novel findings might be worthy pursuing for validation in larger patient series. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-15.

Effect of Margin Width on Local Recurrence in Triple-Negative Breast Cancer Patients Treated with Breast-Conserving Therapy

The effect of increasing negative margin width after breast-conserving therapy (BCT) on local recurrence (LR) is controversial. LR rates vary by subtype, with the highest rates seen in triple-negative breast cancer (TNBC). This study examined LR rates in relationship to margin width in TNBC treated with BCT. Women with TNBC who underwent BCT between 1999 and 2009 were identified. Margins were defined as positive (ink on tumor), 0.1-2.0, and 2 mm. Patients with positive margins were excluded. Statistical comparisons were by t test, Fisher's exact test, and Wilcoxon rank sum test. Cumulative incidence of LR was compared by competing-risks methodology. Of 535 cancers, 71 had margins ≤2 mm and 464 had margins >2 mm. At a median follow-up of 84 months (range 8-165 months), there were 37 local, 18 regional, and 77 distant recurrences or deaths as first events. Ten patients had a locoregional recurrence before planned radiotherapy and were excluded from cumulative incidence analyses. The cumulative incidence of LR at 60 months for margins ≤2 mm was 4.7 % (95 % confidence interval 0-10.0) and for >2 mm was 3.7 % (1.8, 5.5) (p = 0.11). After controlling for chemotherapy and tumor size, there was no difference in LR between the two margin groups (p = 0.06). A difference in the risk of distant recurrence or death was not observed (p = 0.53). Margin width of >2 mm was not associated with reduced LR rates. These data support a negative margin definition of no ink on tumor, even in this high-risk TNBC cohort.

Radiotherapy Can Improve the Disease-Free Survival Rate in Triple-Negative Breast Cancer Patients with T1–T2 Disease and One to Three Positive Lymph Nodes After Mastectomy

Several studies have demonstrated poor locoregional control in patients with triple-negative breast cancer (TNBC), compared with other molecular subtypes of breast cancer. We sought to evaluate whether or not postmastectomy radiotherapy (PMRT) improves locoregional recurrence-free survival (LRFS) and disease-free survival (DFS) outcomes in TNBC patients. Between January 2000 and July 2007, 553 TNBC patients treated with modified radical mastectomy from a single institution were analyzed retrospectively. Patients were categorized into three groups: low risk (stage T1-T2N0), intermediate risk (stage T1-T2N1), and high risk (stage T3-T4 and/or N2-N3). Cox proportional hazards models were used to evaluate the association between PMRT and LRFS and DFS times after adjusting for other clinicopathologic covariates. With a median follow-up of 65 months (range, 1-140 months), 51 patients (9.2%) developed locoregional recurrence and 135 patients (24.4%) experienced disease recurrence. On multivariate analysis, PMRT was associated with significantly longer LRFS and DFS times in the entire cohort. In the intermediate-risk group, PMRT was associated with a longer DFS time but not with the LRFS interval. In the high-risk group, PMRT was associated with significantly longer LRFS and DFS times. PMRT is associated with longer LRFS and DFS times in high-risk TNBC patients and a longer DFS time in intermediate-risk TNBC patients. Prospective randomized studies are needed to investigate the best locoregional treatment approaches for patients with this molecular subtype of breast cancer.