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Adjuvant therapy; Breast cancer; Olaparib Terapia adyuvante; Cáncer de mama; Olaparib Teràpia adjuvant; Càncer de mama; Olaparib Background The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. Patients and methods One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. Results With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI −0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. Conclusion With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals. Funding for this work, which was conducted as a collaborative partnership among the Breast International Group, NRG Oncology, Frontier Science, AstraZeneca, and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, U.S.A. (MSD), was provided by the National Institutes of Health (grant numbers: U10CA 180868, UG1CA 189867, and U10CA 180822) and by AstraZeneca as part of an alliance between AstraZeneca and MSD. Provision of olaparib and placebo was from AstraZeneca.

Supplemental Table 1. Genes and rearrangements studied; Supplemental Table 2: Genes added over time to Guardant360 assay; Supplemental Table 3. Defining concordance and partial concordance for a particular gene; Supplemental Table 4. Concordance and partial concordance among only genes with genomic alterations in either assay; Supplemental Table 5. Sensitivity, specificity, and diagnostic accuracy across 5 genes, including variants of unknown significance (VUS)

Supplemental Table 1: Treatments by subgroup. Supplemental Figure 1A-D: Association of baseline MAF and NOA with survival

Purpose:Circulating tumor DNA (ctDNA) is a promising tool for noninvasive longitudinal monitoring of genomic alterations. We analyzed serial ctDNA to characterize genomic evolution in progressive metastatic breast cancer.Experimental Design:This was a retrospective cohort between 2015 and 2019 obtained under an Institutional Review Board–approved protocol at Northwestern University (Chicago, IL). ctDNA samples were analyzed with Guardant360 next-generation sequencing (NGS) assay. A total of 86 patients had at least two serial ctDNA collections with the second drawn at first post-NGS progression (PN1) by imaging and clinical assessment. A total of 27 participants had ctDNA drawn at second post-NGS clinical progression (PN2). We analyzed alterations, mutant allele frequency (MAF), number of alterations (NOA), and sites of disease on imaging in close proximity to ctDNA evaluation. Matched pairs' variations in MAF, NOA, and alterations at progression were tested through Wilcoxon test. We identified an independent control cohort at Massachusetts General Hospital (Boston, MA) of 63 patients with serial ctDNA sampling and no evidence of progression.Results:We identified 44 hormone receptor–positive, 20 HER2+, and 22 triple-negative breast cancer cases. The significant alterations observed between baseline and PN1 were TP53 (P < 0.0075), PIK3CA (P < 0.0126), AR (P < 0.0126), FGFR1 (P < 0.0455), and ESR1 (P < 0.0143). Paired analyses revealed increased MAF and NOA from baseline to PN1 (P = 0.0026, and P < 0.0001, respectively). When compared with controls without progression, patients with ctDNA collection at times of progression were associated with increased MAF and NOA (P = 0.0042 and P < 0.0001, respectively).Conclusions:Serial ctDNA testing identified resistance alterations and increased NOA and MAF were associated with disease progression. Prospective longitudinal ctDNA evaluation could potentially monitor tumor genomic evolution.

Purpose: Circulating tumor DNA (ctDNA) is a promising tool for noninvasive longitudinal monitoring of genomic alterations. We analyzed serial ctDNA to characterize genomic evolution in progressive metastatic breast cancer. Experimental Design: This was a retrospective cohort between 2015 and 2019 obtained under an Institutional Review Board–approved protocol at Northwestern University (Chicago, IL). ctDNA samples were analyzed with Guardant360 next-generation sequencing (NGS) assay. A total of 86 patients had at least two serial ctDNA collections with the second drawn at first post-NGS progression (PN1) by imaging and clinical assessment. A total of 27 participants had ctDNA drawn at second post-NGS clinical progression (PN2). We analyzed alterations, mutant allele frequency (MAF), number of alterations (NOA), and sites of disease on imaging in close proximity to ctDNA evaluation. Matched pairs' variations in MAF, NOA, and alterations at progression were tested through Wilcoxon test. We identified an independent control cohort at Massachusetts General Hospital (Boston, MA) of 63 patients with serial ctDNA sampling and no evidence of progression. Results: We identified 44 hormone receptor–positive, 20 HER2+, and 22 triple-negative breast cancer cases. The significant alterations observed between baseline and PN1 were TP53 (P < 0.0075), PIK3CA (P < 0.0126), AR (P < 0.0126), FGFR1 (P < 0.0455), and ESR1 (P < 0.0143). Paired analyses revealed increased MAF and NOA from baseline to PN1 (P = 0.0026, and P < 0.0001, respectively). When compared with controls without progression, patients with ctDNA collection at times of progression were associated with increased MAF and NOA (P = 0.0042 and P < 0.0001, respectively). Conclusions: Serial ctDNA testing identified resistance alterations and increased NOA and MAF were associated with disease progression. Prospective longitudinal ctDNA evaluation could potentially monitor tumor genomic evolution.

Introduction: Circulating tumor cells (CTCs) are the roots of metastasis which is the main cause for death in metastatic breast cancer (MBC). CTCs enumeration is strongly prognostic in advanced disease and can stratify patients in two distinct disease, Stage IV aggressive and Stage IV indolent. In the former disease, the detection of CTC clusters and HER2-expression increase prognostic and predictive value. The metastatic cascade is a complex, regulated process involving immune cells and endothelial cells for progression and neoangiogenesis. Circulating endothelial cells (CECs) from the inner wall of blood vessels are shed into the blood stream during formation of blood vessels which is considered a sensitive marker of endothelial damage in pathological conditions such as cancer. CECs have been also studied as a biomarker for tumor progression and monitoring anti-angiogenic therapeutic effects in MBC. We evaluated the concomitant detection of CTCs and CECs in MBC patients, along with expression of HER2 in CTCs that may offer an interesting clue to elucidate the metastasis mechanisms. Methods: Whole blood samples (7.5ml/each) were collected from 14 stage IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS System (Menarini) by using CTC Kit contains specific antibodies targeting the EpCAM for capturing CTCs, anti-CK-PE (for epithelial cells), DAPI (for nucleus), anti-CD45-APC (for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. Meanwhile, the same patients’ blood samples (4.0ml/each) were processed for CEC analyzed by using CEC kit which immunomagnetically captures CD146+ cells, and then stains the cells for CD105-PE (specific for protein endolgin), CD45-APC, nucleus-DAPI. The positive CECs were classified as CD 146+, CD105+, DAPI+ and CD45-. The associations between CTCs, HER2 expression and CECs were evaluated. Results: The average age of patients was 53.1. Subtypes of Luminal, HER2 positive and TNBC were 64.2% 7.2% and 28.6% respectively. Distant metastasis were found in 13 out of 14 patients, including bone (7), liver (5), Lymph nodes (5) and Pleura (2). CTCs were found positive (≥5, Stage IV aggressive) in 5 patients (range: 5-47, mean=24), and HER expression was identified in all 5 of these cases with a range of numbers between 1 and 7 (mean=4.2). The ratios of HER+ CTC/total ratios were 8.51%, 17.95%, 20%, 30.77%, and 33.33%. HER2 expression were defined officially in our lab according to the percentile of positive HER2 CTCs/Total CTCs and the expression intensity as - ( Citation Format: Qiang Zhang, Paolo D'Amico, Jeannine Donahue, Lorenzo Gerratana, Andrew A. Davis, Saya Liz Jacob, Zheng Cai, Elena Vagia, Wenan Qiang, Ami N. Shah, Katy Kerby, Lisa Flaum, Youbin Zhang, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. The detection and enumeration of circulating tumor cells (CTCs) and circulating endothelial cells (CECs) in metastatic breast cancer [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 PS2-06.

Background: Inflammatory breast cancer (IBC) has a distinctive and aggressive clinical behavior but its underlying biological characteristics have not been fully elucidated. The extended analysis of somatic alterations in The Cancer Genome Atlas (TCGA) highlighted canonical oncogenic pathways that were consistently represented across different tumor subtypes. The aim of this study was to translate such pathway-based characterization to the clinical setting through ctDNA NGS to dissect IBC’s biology and prognosis. Methods: The study retrospectively analyzed 255 metastatic breast cancer (MBC) patients (pts) treated and characterized for ctDNA at Northwestern University (Chicago, IL). ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Only non-synonymous alterations were analyzed. Pathway classification was defined based on prior work (Sanchez-Vega F et al, Cell. 2018). Associations among clinical characteristics, pathway classification, and IBC were explored through uni- and multivariate logistic regression; survival was tested though uni- and multivariate Cox regression both for progression-free survival (PFS) and overall survival (OS). Results: Of 255 enrolled pts, 124 (48%) were diagnosed with hormone receptor positive (HR pos) MBC, 75 (30%) with HER2-positive (HER2_pos) MBC and 56 (22%) with triple negative (TNBC) MBC. IBC was diagnosed in 74 pts (30%). Receptor-tyrosine kinase, RTK (130 pts, 51%), p53 (130 pts, 51%), PI3K/Akt (116 pts, 46%), and cell cycle (91 pts, 36%) were the most often altered pathways. The multivariate model highlighted the association of IBC with HER2_pos (OR: 2.19; 95%CI: 1.09 - 4.38; P=0.0276), an increased number of alterations in the p53 pathway (OR: 2.05; 95%CI: 1.12 - 3.75; P=0.0197) and a decreased number of alterations in the RAS pathway (OR: 0.34; 95%CI: 0.14 - 0.80; P=0.0137). Decreased alterations in the ER pathway were borderline significant (OR: 0.48; 95%CI: 0.22 - 1.03; P=0.0584). Only cell cycle alterations had an impact on PFS for IBC (HR: 2.20; 95%CI: 1.18 - 4.08; P=0.0127), while p53 and Wnt had an impact on nonIBC (respectively HR: 2.00; 95%CI: 1.23 - 3.25; P=0.0052 and HR: 3.40; 95%CI: 1.20 - 9.64; P=0.0212). The univariate model showed a significant impact on OS RAF, ER, and cell cycle pathways alterations for IBC, the role of ER and cell cycle pathways alterations was confirmed in the multivariate model (respectively HR: 6.19; 95%CI: 1.63 - 23.48; P=0.0073 and HR: 3.79; 95%CI: 1.04 - 13.75; P=0.0431). The multivariate model showed a prognostic impact only for p53 in the nonIBC subgroup (HR: 2.20; 95%CI: 1.11 - 4.36; P=0.0237) Conclusion: The ctDNA-based oncogenic signaling pathway characterization showed different biological and prognostic features across IBC and nonIBC MBC patients. Alterations of the p53 pathway were more likely to be present in IBC pts, while alterations in the RAS pathway were less represented in this cohort. ER and cell cycle pathways’ alterations impacted the OS of IBC MBC patients. Although preliminary, these results suggest a more comprehensive biological characterization based on ctDNA for treatment selection and clinical decision-making. Main alterations and pathwayGeneNumber of alterations%PathwayTP5318815.58p53PIK3CA14111.68PI3K/AktERBB2725.97RTKESR1705.8ERMYC574.72MycFGFR1463.81RTKEGFR453.73RTKCCNE1383.15Cell cycleMET322.65RTKNF1322.65RAS Citation Format: Lorenzo Gerratana, Andrew A Davis, Marco Mina, Saya L Jacob, Qiang Zhang, Ami N Shah, Paolo D’Amico, Neelima Katam, Firas Wehbe, Elena Vagia, Lisa Flaum, Leonidas Platanias, Amir Behdad, Fabio Puglisi, William J Gradishar, Giovanni Ciriello, Massimo Cristofanilli. Investigating oncogenic signaling pathways in inflammatory metastatic breast cancer (MBC) though circulating tumor DNA (ctDNA) next-generation sequencing (NGS) [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 PS2-33.

Background: Circulating tumor DNA (ctDNA) has emerged as a potential tool for detecting disease recurrence, monitoring response to therapy, and identifying resistance mutations in the peripheral blood. With increased frequency of testing, there is an unmet need to recognize putative germline variants in ctDNA, and the probability that these variants are associated with inherited conditions. Here, we evaluated a large cohort of breast cancer patients who underwent ctDNA evaluation to determine the type and frequency of ctDNA mutations identified with confirmed germline testing. Methods: We reviewed ctDNA testing from a single institution (Northwestern University). All breast cancer patients who had next-generation sequencing (NGS) performed by Guardant Health (Redwood City, CA) from 2015-2020 were included in this retrospective study. An allele frequency cutoff of 30% was pre-established as a threshold to review patient charts to determine whether genetic counseling and germline testing were performed, along with the timeframe of this testing (e.g. before or after ctDNA evaluation). Clinical information including demographics, pathology, tissue NGS testing, and germline testing were collected. Descriptive analyses and statistical associations were performed using STATA. Results: The initial cohort consisted of 520 patients with breast cancer who underwent ctDNA testing. From this, we identified 84 patients (16.2%) who had at least one variant with allele frequency ≥30%. The most common variants identified were the following: TP53 (34%), PIK3CA (27%), BRCA1 (9%), BRCA2 (8%), and AKT1 (4%). Guardant360 classified 99% of these variants as pathogenic and 1% as a variant of unknown significance. Germline positivity using a separate CLIA-approved test for this indication was confirmed at the following frequencies: BRCA1 (2 of 8 positive, 25%), BRCA2 (2 of 5 positive, 40%), PIK3CA (0 of 5 positive), and TP53 (0 of 26 positive). In total, 14% of patients with ctDNA allele frequency ≥30% had a confirmed germline mutation. Lower age at breast cancer diagnosis was significantly associated with the probability of germline testing prior to ctDNA evaluation (P=0.0001). For patients who had a variant with allele frequency ≥30%, 24.3% never received genetic counseling or germline testing. Conclusion: High allele frequency ctDNA variants (≥30%) were present in 16% of patients who underwent ctDNA evaluation with 14% of these variants confirmed as true germline variants. Consenting patients for ctDNA testing should include the possibility of identifying putative germline variants, and criteria should be established to refer patients for subsequent genetic counseling and germline testing, given the potential implications for patients and their family members. Citation Format: Andrew A Davis, Michael C Burns, Lorenzo Gerratana, Paolo D'Amico, Saya Jacob, Ami Shah, Neelima Katam, Firas Wehbe, Qiang Zhang, Elena Vagia, Lisa Flaum, Kalliopi P. Siziopikou, Leonidas C Platanias, Amir Behdad, William J Gradishar, Massimo Cristofanilli. Identification of incidental putative germline variants in circulating tumor DNA [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 PS2-08.

Introduction: Prognosis of metastatic breast cancer (MBC) is initially predicted by the cancer’s characteristics based on AJCC TNM system, including the size of the cancer tumor, invasion into nearby tissue, lymph nodes and other parts of the body beyond the breast. Although additional information including hormone-receptor status, HER2 status, and possibly Oncotype DX score contributed to improve prognostic evaluation, predicting clinical outcomes and treatment benefit for MBC is still a challenge in clinic because of the clinical and biologically heterogeneous condition. We recently reported that CTCs enumeration can classify MBC in two distinct prognostic groups independently of clinical and molecular characteristics (Crit Rev Oncol Hematol. 2019). Moreover, our group reported that CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor (2019 AACR #1919). Here we compared CTCs enumeration of Stage III and Stage IV, which would be helpful to evaluate the MBC metastasis capability and treatment in clinic. Methods: The study included 38 specimens prospectively collected under IRB-approved protocol from 38 patients with Stage III MBC, and 254 specimens from 254 patients with stage IV MBC who received standard systemic treatments based on disease subtypes at NMH (2016-2020). Duplicate whole blood samples (7.5ml/each) were collected in EDTA tubes from these patients who were longitudinally characterized for CTCs before therapy (baseline). CTCs enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Menarini) by using CELLSEARCH® CXC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI for staining the cell nucleus, anti-CD45-APC is specific for leukocytes (2019 ASCO #1036). The CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. Kruskal-Wallis test was used for statistics. Results: Patients were classified as Luminal, HER2 positive and TNBC disease subtypes in 46.6%, 46.7% and 6.7% respectively in Stage III patients, and 54%, 18% and 28% respectively in Stage IV patients. The patients at age above 50 were 26.% in Stage III group and 68% in Stage IV group respectively. IBC patients represented 61.5% and 33.5% of Stage III and Stage IV patients respectively. Metastasis in liver, lung and bone were diagnosed in 40.7%, 40.2% and 62.8% in Stage IV patients. CTC negative ( Citation Format: Qiang Zhang, Zheng Cai, Lorenzo Gerratana, Paolo D'Amico, Andrew A. Davis, Saya Liz Jacob, Elena Vagia, Ami N Shah, Lisa Flaum, Youbin Zhang, Wenan Qiang, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Prognostic value of baseline circulating tumor cells (CTCs) enumerations is for stage III and stage IV breast cancer [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 PS2-20.

Introduction: Although CTCs display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. A portion of CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of forming distant metastasis in MBC as compared to individual CTCs (Aceto N. Cell, 2015). However, genomic characterization of CTCs-clusters compared to single CTCs remain largely unknown. We previously reported single CTC sequencing for HER2+ CTCs (2020 AACR #3120). Herein, we report a new finding of heterogeneity profiling for CTC-clusters compared to single CTCs, which would be helpful to evaluate the MBC metastasis capability and treatment in clinic. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV MBC patients before therapy. CTC enumeration was performed using the FDA-cleared CellSearch™ System (Menarini) targeting the EpCAM antigen for capturing CTCs which were then stained by Anti-CK-PE, DAPI, anti-CD45-APC and anti-HER2-FITC. The CTC-clusters and single CTCs were isolated using DEPArrayTM System (Menarini). DNA was isolated from CTC-clusters and single CTCs by ArcturusTM PicoPureTM DNA Extraction kit. The initial library was prepared by SMARTer® PicoPLEX® Gold Single Cell DNA-Seq Kit, and the exome capture was performed by Twist Human Core Exome EF Multiplex Complete Kit. The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). It was a paired end run, 75×75 bps run with dual indexing. Results: We identified 107 CTCs by CellSearch™, including 93 single CTCs, 14 CTC-clusters and 145 WBCs. Autologous CTC-clusters (CK+DAPI+CD45-, Group 1), single CTCs (CK+DAPI+CD45-, Group 2), and leukocytes (CK-DAPI+CD45+, Group 3) were sequenced respectively. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 60,638 counts (6.77%) and 70,334 counts (8.20%) for exon variants in CTC-clusters and single CTCs respectively, 507,595 counts (56.69%) and 486,119 counts (56.69%) for intron variants, 194,026 (21.67%) and 175,819 counts (20.51%) for intergenic variants, 54,174 counts (6.05%) and 50,370 counts (5.87%) for downstream genes, 51,716 counts (5.78%) and 45,915 counts (5.36%) for upstream genes, and 3.04% and 3.37% of others variants in CTC-clusters and single CTCs respectively. Meanwhile, there was 0 count for exon and intron variants found in Group 3. There were 60 and 79 gene variants (SNP and Ins-Del) identified to have the highest impact effect (≥20) on CTC-clusters and single CTC exons respectively, which affect significantly on the functional proteins coding. Among the top 50 high impact gene variants in each group, there were 25 gene alteration sites were similar in Group 1 and 2, including XYLB, RAN, QPCT, HPGDS, HDAC8, GABBR2, CYP11B2 and CHKA. Specific to Group 1, there were 25 gene alterations which were primarily related to cellular proliferation and tumor promotion (AMD1), liver drug clearance (CES1), tissue remodeling (CHI3L1), immune cytokine signaling (JAK1) and metabolism (ASRGL1). Meanwhile, there are 25 specific gene alterations in Group 2 compared to Group 1, which were associated with nucleotide-excision repair (DDB1 and FAN1) chromosome positioning (KIF11), cell growth, differentiation, mitotic cycle, oncogenic transformation (PTPN3 and MAPK14), apoptosis (CASP1) and cell growth (CTNNB1). Conclusion: Genomic characterization of CTC-clusters compared to autologous single CTCs and leukocytes elucidated new specific gene alterations in CTC-clusters associated with most aggressive disease metastasis in MBC, which will help to gain new insights on the molecular mechanisms associated with the metastasis and find new molecularly driven therapies for disease metastasis. Citation Format: Qiang Zhang, Lorenzo Gerratana, Paolo D'Amico, Andrew A. Davis, Saya Liz Jacob, Xinkun Wang, Zhe Ji, Zheng Cai, Elena Vagia, Wenan Qiang, Ami Shah, Youbin Zhang, Lisa Flaum, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Genetic profiling for circulating tumor cell clusters to unveil molecular drivers of metastasis [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 PS2-05.

Background: As the role of precision medicine in metastatic breast cancer (MBC) expands, there is an increasing emphasis on diagnostic tools to characterize both somatic and germline alterations that drive carcinogenesis. Circulating tumor DNA (ctDNA) has provided an important means for real-time, non-invasive detection and monitoring of tumor somatic alterations without standard paired white blood cell testing. However, its application for the detection of germline mutations remains relatively understudied. Here, we characterize the role of BRCA1&2 ctDNA mutant allele frequency (MAF) to identify underlying putative germline BRCA1&2 mutations. Methods: Patient data were retrospectively obtained under an IRB-approved protocol to review ctDNA data at Northwestern University between 2015 and 2020. All ctDNA samples were analyzed using the Guardant360 next-generation sequencing (NGS) assay (Guardant Health). Patients with ctDNA alterations in the BRCA genes were identified at all mutant allele frequencies (MAF). Clinical data were collected including breast cancer subtype, prior lines of therapy, tissue-based NGS and germline testing information. Separate CLIA-approved testing performed per standard of care was used as the gold standard for germline testing. Statistical analysis was used to test the association of MAF cut-offs with the presence or absence of a germline alteration. Receiver operating characteristic (ROC) analysis was performed. Results: We identified 127 patients with breast cancer who underwent ctDNA collection. There were 69 HR+ HER2-, 24 HER2+, and 34 triple negative breast cancer (TN) patients. Of these, 8 patients had known BRCA1 germline mutations, as confirmed with separate germline testing, and 9 patients had known BRCA2 germline mutations. BRCA1 germline mutations were significantly association with age < 40 (p=0.016) and triple negative subtype (p=0.042). Mean ctDNA BRCA1 MAF was 2.27% (Standard Deviation {SD} 10.19%) and mean BRCA2 MAF was 2.04% (SD 9.51%). BRCA1/2 MAF was analyzed with respect to germline mutations through ROC analysis. For BRCA1 ctDNA MAF cut-off 32.4% was associated with confirmed putative germline mutation. This cutoff demonstrated sensitivity of 1 and specificity of 0.99. Area under the curve (AUC) was 1.00 at this cut-off. For BRCA2, ctDNA MAF cutoff of 28.5% was associated with confirmed putative germline mutations. This cutoff demonstrated sensitivity of 0.86, specificity of 0.99 and AUC of 0.93. Discussion: ctDNA alterations of BRCA1 with MAF>32.4% and BRCA2 with MAF>28.5% in clinical ctDNA testing were useful criterion to identify germline BRCA mutations. The data suggest the potential to utilize ctDNA as a diagnostic tool to predict germline mutations based on MAF thresholds. Future prospective studies are needed to determine how ctDNA testing may be incorporated into existing clinical algorithms to refer patients for germline testing. Citation Format: Saya Jacob, Andrew Davis, Paolo D'Amico, Lorenzo Gerratana, Michael Burns, Ami Shah, Neelima Katam, Firas Wehbe, Qiang Zhang, Elena Vagia, Lisa Flaum, Kalliopi Siziopikou, Leonidas Platanias, Amir Behdad, William Gradishar, Massimo Cristofanilli. Circulating tumor DNA (ctDNA) as a diagnostic tool to identify putative germline BRCA mutations [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 PS2-10.

PURPOSE Patients with triple-negative breast cancer (TNBC) and residual invasive disease (RD) after completion of neoadjuvant chemotherapy (NAC) have a high-risk for recurrence, which is reduced by adjuvant capecitabine. Preclinical models support the use of platinum agents in the TNBC basal subtype. The EA1131 trial hypothesized that invasive disease-free survival (iDFS) would not be inferior but improved in patients with basal subtype TNBC treated with adjuvant platinum compared with capecitabine. PATIENTS AND METHODS Patients with clinical stage II or III TNBC with ≥ 1 cm RD in the breast post-NAC were randomly assigned to receive platinum (carboplatin or cisplatin) once every 3 weeks for four cycles or capecitabine 14 out of 21 days every 3 weeks for six cycles. TNBC subtype (basal v nonbasal) was determined by PAM50 in the residual disease. A noninferiority design with superiority alternative was chosen, assuming a 4-year iDFS of 67% with capecitabine. RESULTS Four hundred ten of planned 775 participants were randomly assigned to platinum or capecitabine between 2015 and 2021. After median follow-up of 20 months and 120 iDFS events (61% of full information) in the 308 (78%) patients with basal subtype TNBC, the 3-year iDFS for platinum was 42% (95% CI, 30 to 53) versus 49% (95% CI, 39 to 59) for capecitabine. Grade 3 and 4 toxicities were more common with platinum agents. The Data and Safety Monitoring Committee recommended stopping the trial as it was unlikely that further follow-up would show noninferiority or superiority of platinum. CONCLUSION Platinum agents do not improve outcomes in patients with basal subtype TNBC RD post-NAC and are associated with more severe toxicity when compared with capecitabine. Participants had a lower than expected 3-year iDFS regardless of study treatment, highlighting the need for better therapies in this high-risk population.

PURPOSE Liquid biopsy–based biomarkers, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), are increasingly important for the characterization of metastatic breast cancer (MBC). The aim of the study was to explore CTCs and ctDNA dynamics to better understand their potentially complementary role in describing MBC. METHODS The study retrospectively analyzed 107 patients with MBC characterized with paired CTCs and ctDNA assessments and a second prospective cohort, which enrolled 48 patients with MBC. CTCs were immunomagnetically isolated and ctDNA was quantified and then characterized through next-generation sequencing in the retrospective cohort and droplet digital polymerase chain reaction in the prospective cohort. Matched pairs variations at baseline, at evaluation one (EV1), and at progression were tested through the Wilcoxon test. The prognostic role of ctDNA parameters was also investigated. RESULTS Mutant allele frequency (MAF) had a significant decrease between baseline and EV1 and a significant increase between EV1 and progression. Number of detected alterations steadily increased across timepoints, CTCs enumeration (nCTCs) significantly increased only between EV1 and progression. MAF dynamics across the main altered genes was then investigated. Plasma DNA yield did not vary across timepoints both in the retrospective cohort and in the prospective cohort, while the short fragments fraction showed a potential role as a prognostic biomarker. CONCLUSION nCTCs and ctDNA provide complementary information about prognosis and treatment benefit. Although nCTCs appeared to assess tumor biology rather than tumor burden, MAF may be a promising biomarker for the dynamic assessment of treatment response and resistance.

Introduction: Although circulating tumor cells (CTC) display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. ESR1 mutations are a major mechanism of acquired endocrine resistance in metastatic breast cancer (MBC). We previously reported that ESR1 mutations in circulating tumor DNA (ctDNA) was associated with worse prognosis in MBC (2020 ASCO). Herein, we report a new ultra-high sensitivity approach of ESR1 mutations assay for single CTC by using DEPArray system and Droplet Digital PCR (ddPCR). Methods: Whole blood sample (7.5ml/each) was collected from stage IV MBC patients and then CTC enumeration was performed in CellSearch™ System by targeting the EpCAM antigen (2020 AACR #3120). The single CTC and single white blood cell (WBC) were then isolated from CellSearch cartridges by using DEPArray࣪ System (Menarini). The single CTC DNA was isolated by QIAamp DNA Micro Kit (Qiagen). ESR1 mutations hotspots including p.E380Q, p.Y537S and p.D538G were analyzed by using QX200™ ddPCR System (Annealing/Extension=58 °C, 40 cycles) and the corresponding probes (Mutation-FAM and Wild type-HEX mixed) for p.E380Q-dHsaMDS500014536, p.Y537-dHsaMDS975379796, p.D538G- dHsaMDS460485301 respectively (Bio-Rad). Meanwhile, corresponding plasma ctDNA was analyzed by Guardant 360 Health NGS-based assay for a 73 genes panel for single nucleotide variants. Results: Positive controls (using 0.00000325ng pure positive DNA) and negative control (using water) were performed in each ddPCR assay. In cohort 1: total of 8 samples including 4 single CTC samples and 4 single WBC samples were collected using the DEPArray system. Wild type ESR1 (p.E380Q, p.Y537S and p.D538G) was detected in all 4 single CTC samples and 4 single WBC samples. There were 3 copies, 3.4 copies, 1.6 copies and 1.8 copies of p.E380Q mutation droplets found in 4 single CTC samples when there were no p.Y537S or p.D538G mutations found in these samples. There was no ESR1 mutation found in the corresponding ctDNA. In cohort 2: Two samples including 4 CTCs and 100 CTCs were collected from CellSearch cartridges. There were 0.09 vs 1 copies (8.25%) and 0.1 vs 0.31 copies (24.39%) of p.E380Q mutation droplets found in these two samples respectively, compared to ctDNA results of 1.80% and 0 respectively. Furthermore, there was 0.08 vs 0.67 copies (10.66%) of p.D538G mutation droplets were found in the sample including 100 CTCs compared to 12.70% mutations detected in the corresponding ctDNA. Conclusion: Our new approach verified the feasibility of ESR1 mutations assay in very low amount DNA from single CTCs with high sensitivity even when ESR1 mutation could be found in CTCs but not ctDNA. This approach may offer a strong evidence for early disease metastasis, efficacy of individualized treatment monitoring and personalizing cancer therapy for precision medicine. Citation Format: Qiang Zhang, Paolo D'Amico, Marwa Manai, Carolina Reduzzi, Andrew A. Davis, Lorenzo Gerratana, Weijun Qin, Jianhua Jiao, Saya L. Jacob, Jeannine Donahue, Youbin Zhang, Lisa Flaum, Massimo Cristofanilli, Leonidas C. Platanias, Ami N. Shah, William Gradishar. A novel ESR1 mutation assay for single circulating tumor cell by application of DEPArray࣪ System and Digital Droplet PCR [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 1950.

Introduction: CTCs play a critical role in breast cancer (BCa) metastases. Rapid identification of live CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic disease. However, CTCs are present at very low concentrations in the peripheral blood, and CTC enumeration is costly and technically challenging. We previously reported that cell-size based device utilizing a microfluidic cassette could be used to isolate live blood cells (Zhang et al. 2019 AACR). Here we describe a pilot study using novel microfluidic Parsortix workflow to identify live CTCs for patients with Stage IV BCa. Methods: Four whole blood samples (7.5ml/each) were collected from each of 10 patients with stage IV BCa patients who had continued clinical progression of their disease on systemic therapy, under a Northwestern University IRB-approved study (NU20B03). All samples were stored at 15-30 °C in EDTA tubes. Live CTCs enrichment was performed using a cell size and deformability based Parsortix system (Angle) which utilizes a microfluidic based disposable cassette containing a step separation structure. The four samples from each patient were loaded onto the device and the separation procedure was started by using 4.5μm, 6.5μm 8μm and 10μm separation cassettes, respectively. After separation and cleaning, the CTCs captured by the Parsortix system were harvested by inverting the flow direction and then flushing from the cassette in 200μL PBS. The harvested CTCs were multi-stained directly by fluorescence labeled antibodies for Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes) (Menarini). Flow cytometry (FCM) was used to identify the CTCs which were classified as CK+, DAPI+ and CD45-. White blood cells from patient samples and the human breast cancer cell line MDA-MB-231 were used as negative and positive controls, respectively. Results: A total of 500 and 200 MDA-MB-231 cells were processed as controls, which were collected completely by Parsortix and identified by FCM confirming that Parsortix system could recover the CTCs effectively. CTCs were classified based on morphology and phenotype as CK+, DAPI+ and CD45-. Live CTCs (≥1) were detected in 8 out of 10 (80%), 9 out of 10 (90%), 7 out of 10 (70%) and 5 out of 10 (50%) samples by using 4.5μm (Group 1), 6.5μm (Group 2), 8μm (Group 3) and 10μm (Group 4) separation cassettes. Maximum CTCs isolated from each group were 18, 32, 12 and 8 from Group 1, 2, 3 and 4, respectively. The corresponding average CTCs isolated from each sample were 3.5 CTCs, 5.6 CTCs, 2.5 CTCs and 1.7 CTCs from Group 1, 2, 3, and 4, respectively. The 6.5μm cassettes (Group 2) using for CTCs collection had significantly higher efficacy on collection of CTCs compared to other three groups. Conclusions: In this pilot study, our findings demonstrated that the cell-sized dependent Parsortix system using multiple staining and FCM is an effective and specific approach for rapid recovery and identification of CTCs from Stage IV BCa patients. With further optimization, this strategy can help to accurately evaluate CTCs and offers a potential technique for diagnosis and treatmentmonitoring of patients with metastatic breast cancer. Citation Format: Qiang Zhang, Marwa Manai, Andrew Davis, Carolina Reduzzi, Paolo D’Amico, Saya Liz Jacob, Jianhua Jiao, Weijun Qin, Lisa Flaum, Amir Behdad, Massimo Cristofanilli, Ami Shad, Leonidas Platanias, William Gradishar. Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry [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 LB117.

Background: We describe the genomic landscape of circulating tumour DNA (ctDNA) across pathological subtypes of metastatic breast cancer. Methods: 255 clinically annotated patients with ctDNA testing by Guardant360 were stratified into HR+, HER2+, and TNBC cohorts. Frequency and heterogeneity of alterations were reported. Paired ctDNA and tissue sequencing were compared for a subset of patients. The association of ctDNA and metastatic sites of disease on imaging was also assessed. Findings: 89% of patients had at least one ctDNA alteration detected. The most common single nucleotide variants (SNVs) for HR+ patients were PIK3CA, ESR1, and TP53. For HER2+, these were TP53, PIK3CA, and ERBB2 with ERBB2 as the most frequent copy number variant (CNV). For TNBC, the most common SNVs were TP53 and PIK3CA, and the most frequent CNVs were MYC, CCNE1, and PIK3CA. TNBC patients had a significantly higher mutant allele frequency (MAF) of the highest variant compared to HR+ or HER2+ patients (P

Circulating tumor DNA (ctDNA) is gaining momentum as sensitive diagnostic tool for advanced disease characterization because of its ability both to capture the tumor's heterogeneity and its dynamic adaptations. However, the consistency between all the available platforms is still debated. The aim of the study was to explore the performance of the novel diagnostic NGS platform PredicinePLUS™ and to compare its results with the clinically available Guardant360™ platform for possible analytical inconsistencies. The study suggests that PredicinePLUS™ NGS platform can detect genomic alterations and measure allele frequency in samples of MBC patients and confirmed that different NGS platforms could be potentially compared provided that certain sample management and analytical requirements are met.

Introduction: Metastatic breast cancer (MBC) is a challenging clinical condition because most tumors will develop resistance to endocrine treatment as the cancer progresses. We previously reported the correlation of ESR1 mutations in circulating tumor DNA (ctDNA) and CTCs resulted in worse prognosis in MBC (ASCO-2020). In this study, we report a new finding that specific hotspots of ESR1 ctDNA mutations are associated with endocrine therapy resistance status in MBC and their allele frequency (AF) can be used to monitor response. . Methods: Plasma cfDNA were collected from a total of 299 MBC patients (2016-2019) after informed consent under an IRB-approved trial at the Lurie Cancer Center at Northwestern University. ER+ patients are 180 out of 297 (61%). Patients with HR+ MBC were evaluated before endocrine therapy (Anastrozole, Exemestane and Letrozole ) +/- CDK4/6 inhibitors (Palbociclib, Ribociclib, Abemaciclib), and every six months after initiation of therapy. Response to therapy was recorded according to the different time points. Endocrine resistance is defined as relapse during the first 2 years of adjuvant endocrine therapy or progressive disease within the first 6 months of first-line endocrine therapy for metastatic breast cancer. Plasma ctDNA was isolated using a Qiagen circulating nucleic acid kit, and then was analyzed using the Guardant360 Health next-generation sequencing (NGS)-based assay (Guardant Health). In this study, only patients with ESR1 mutations were included for statistical analyses of correlation between the hotspots mutation with the endocrine therapy resistance by using Causal Inference approach. All statistical analyses were conducted Mann-Whitney U test by IBM SPSS version 23.0. Results: There were total of 239 ESR1 ctDNA mutations detected in 18 hotspots from 42 patients at different time points, among which there were 131 endocrine therapy resistance status in all time points. The numbers of ESR1 hotspots mutations and the corresponding endocrine therapy resistance cases were found in the following hotspots respectively as Q314 (3 cases with mutation, 0 cases with resistance), T347T (4, 0), N359I (4, 0), K362N (4, 0), E380Q(32, 16), V392I (1, 1), G442R(3, 0), F461I(3, 0), S463P (3, 2), S464 (1, 0), I487M(14, 0), K520K (2, 0), M528V (5, 0), L536H (31, 19), Y537N/C/S (46, 34), D538G (77, 59), D545D(2, 0) and Q565 (4, 0). There were 50%, 61.2%, 73.9% and 76.6% patients developed endocrine therapy resistance when they had E380Q, L536H, Y537N/C/S and D538G mutations respectively at any time points. Furthermore, among these 4 hotspot mutations, the MBC patients with lack of response to endocrine therapy have significantly higher percentage of mutated DNA than the patients with sensitive response. For the patients with E380Q mutation, the mean percentage of mutation DNA was 8.44% for those with lack of response and lower than 0.01% for those with sensitive response (P Citation Format: Qiang Zhang, Paolo D'Amico, Weijun Qin, Jianhua Jiao, Andrew A. Davis, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N. Shah, Amir Behdad, Lisa Flaum, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Esr1 hotspot mutations in circulating tumor DNA mutation are associated with endocrine therapy resistance in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-04.

Introduction: CTCs are tumor cells that circulate in the blood of patient with primary and MBC and, are responsible for seeing of metastasis. The monitoring of CTCs in MBC emerged as strong prognostic and possible predictive biomarker in oncology over the past couple of decades. Meanwhile, overexpression of HER2 protein has been associated with rapid cell division and worse prognosis of MBC. Here we report a significant correlation between dynamic CTCs enumeration and CTCs-HER2 expression during the systemic therapies, with potential implication to understand treatment resistance. Methods: A total of 298 whole blood samples (7.5ml/each) were collected from 149 patients with stage IV breast cancer (2016-2020) at the Northwestern University Robert H Lurie Comprehensive Cancer Center, before (Baseline) and 3 months after (Time point 2) initiation of systemic treatment. CTC enumerations were performed using the FDA approved CellSearch™ system (Menarini) which is specific for the intracellular protein cytokeratin (CK) in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. We developed a criteria for evaluation of HER2 expression by 4 different categories (0,1+,2+,3+) based on expression intensity in our lab (present in 2021 ASCO). In this study we included CTCs with all intensities of HER2 expression (1+ to 3+) which was standardized in our lab. Mann-Whitney U test was used for statistics. Results: Of the 149 baseline samples, CTC≥1 were found in 101 patients (67.8%). A change in CTCs between baseline and time point 2 for these 101 patients: Three groups were identified: Group 1: 33patients (33%) with increase CTCs; Group 2: 64 patients (63%) with decreased CTC; Group 3: 4 patients (4%) with no change The median increase of total CTCs and HER2+ CTCs in Group 1 were 7.0 and 2.0, respectively; the median decrease of total CTCs and HER2+ CTCs in Group 2 were 9.5 and 2.0, respectively. The change of HER2+ CTCs was significantly correlated with the change of total CTCs after systemic therapy, with the correlation coefficient as rs=0.662 (p Citation Format: Qiang Zhang, Weijun Qin, Paolo D'Amico, Andrew A. Davis, Jianhua Jiao, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N Shah, Amir Behdad, Lisa Flaum, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Dynamic circulating tumor cell changes in enumeration and HER2 expression during systemic therapy for metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-05.

Background: ILC is the second most common type of breast cancer and accounts for approximately 10% of all invasive breast cancers. A hallmark of ILC is the lack of E-cadherin (CDH1) expression, which is frequently used to discriminate between lesions with borderline ductal and lobular histologies. While the genomic landscape of primary ILCs has been well described, less is known about patients (pts) with metastatic-ILC (mILC). Better characterization of the genomic landscape associated with mILC is critical for identifying biomarkers that may provide new insight into ILC tumor biology and ultimately improve long-term outcomes in pts with mILC. Methods: We retrospectively analyzed de-identified next-generation sequencing (NGS) data from 150 advanced/metastatic pts with ILC and 51 with mixed lobular/ductal histology, defined using the histology of the sequenced biopsy. Diagnoses were abstracted from pathology reports submitted at the time of sequencing. We used the stage documented closest in time to biopsy collection, and samples were excluded if the staging date was unknown or exceeded 180 days after the biopsy date. Our dataset consisted of samples that were molecularly profiled using the Tempus xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage, full-transcriptome RNA-seq). The mutations identified for this study include somatic single-nucleotide variants and insertions/deletions. Furthermore, we examined the co-mutational landscape of CDH1-mutant disease and investigated transcript-level expression variation. Results: Mutations in CDH1 occurred in 65.3% of all mILC samples (98/150). CDH1 expression was similar between CDH1-mutant and WT mILC samples (Wilcoxon rank sum test, p=0.8). The median tumor mutational burden (TMB) score was significantly higher in CDH1-mutant samples (Wilcoxon rank-sum test, p=0.010). CDH1-mutant samples were more likely to have a high TMB (≥10 mutations/MB) when compared with the wild-type CDH1 cohort (10% vs. 6.2%), but this difference was not statistically significant (Fisher’s Exact test, p=0.5). Additionally, we observed that the ER+ subtype was more frequent in CDH1-mutant samples, although this difference was not statistically significant (97% vs 88%; Fisher’s exact test, p=0.063). PIK3CA mutations were enriched in CDH1-mutant mILC (Table 1). TBX3 and NCOR1 mutations were also mildly enriched in CDH1-mutant mILC, but these results were not significant when correcting for multiple testing (Table 1). CDH1-mutant mixed histology pts had lower CDH1 expression than WT pts (p Table 1.Frequency of co-mutations in CDH1-mutant vs. WT mILC cohortsGenesCDH1-mutant (n=98)CDH1 WT (n=52)p-value1q-value2n (%)n (%)PIK3CA53 (54%)6 (12%) Citation Format: Andrew A Davis, Amir Behdad, Kayla Viets Layng, Firas Wehbe, Lorenzo Gerratana, Elizabeth Mauer, Alex Barrett, Ami N Shah, Paolo D’Amico, Lisa Flaum, William J Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-01.

Introduction: Estrogen Receptor (ER)-positive breast cancer is the most frequent breast cancer subtype. Endocrine therapy (ET) targeting the ER pathway including CDK4/6 inhibitors represents the main initial therapeutic approach. However, clinical resistance associated with progression of disease caused by ESR1 mutations is a recognized an important mechanism of ET resistance. ESR1 mutations, most often detected from liquid biopsies, have been consistently associated with a worse outcome and are being currently evaluated as a potential biomarker to guide therapeutic decisions. Here we reported a new finding on the association between ESR1 amino acid Y537 mutations in ctDNA and circulating tumor cells (CTC) in metastatic breast cancer (MBC). Methods: The study included 158 ER positive MBC patients enrolled under an IRB-approved trial (NU16B06) at Lurie Cancer Center, Northwestern University. The patients received systemic treatments in 2016-2020. Whole blood samples (7.5ml/each) were collected in EDTA tubes from patients who were longitudinally characterized for CTCs before therapy (baseline). CTCs enumeration were performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CXC Kit contains antibodies targeting EpCAM antigen for capturing CTCs, anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI for staining the cell nucleus, anti-CD45-APC is specific for leukocytes. The CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. Plasma ctDNA was isolated using a Qiagen circulating nucleic acid kit, and then was analyzed using the Guardant360 next-generation sequencing (NGS)-based assay. In this study, only patients with ESR1 mutations were included for statistical analyses of correlation between the hotspot mutation with the endocrine therapy resistance by using Causal Inference approach. All statistical analyses were conducted Mann-Whitney U test by IBM SPSS version 23.0. Results: ESR1 mutations were detected in 40 out of 158 patients at baseline. ESR1 Y537(N/C/S) mutations were detected in 13 patients, among which there were 9 patients who only had the Y537(N/C/S) mutations and no other ESR1 hotspot mutations (Group 1) and there were 4 patients who had polyclonal ESR1 mutations. In the latter cohort, there were 17 ESR1 amino acid mutations detected in 31 patients (Group 2) including Q314, T347T, N359I, K362N, E380Q, V392I, G442R, F461I, S463P, S464, I487M, K520K, M528V, L536H, D538G, D545D and Q565. There were no any ESR1 mutations detected in the other 118 patients (Group 3). The CTC>5/7.5mL (Stage IV aggressive) were found in 66.7%, 38.7% and 29.3% patients in Group 1, Group 2 and Group 3 respectively. The median total CTCs was 26.0/7.5mL in MBC patients with ESR1 Y537(N/C/S) mutations (Group 1). The median of total CTCs was 2.0/7.5mL in the MBC patients with polyclonal ESR1 mutations but not on Y537 (N/C/S) (Group 2), and it was 1.0/7.5mL in the group of metastatic breast cancer without ESR1 mutations (Group 3). The median total CTCs in Group 1 was significantly higher than Group 2 (Mann-Whitney U, P Citation Format: Qiang Zhang, Jianhua Jiao, Paolo D'Amico, Andrew A. Davis, Weijun Qin, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N. Shah, Lisa Flaum, William Gradishar, Leonidas C. Platanias, Massimo Cristofanilli. Esr1 Y537 mutations are associated with increased baseline circulating tumor cells enumeration for patients with estrogen receptor positive metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-08.

While identifying genomic alterations in tumor tissue is the current gold-standard technique for molecular profiling, circulating tumor DNA (ctDNA) represents a noninvasive method of assessing genomic alterations using peripheral blood. The concordance of genomic alterations between two commercially available ctDNA and tissue biopsies was compared in 45 patients with breast cancer using paired next-generation sequencing tissue and ctDNA biopsies. Across all genes, concordance between the two platforms was 91.0% to 94.2%. When only considering genomic alterations in either assay (e.g., excluding wild type/wild type genes), concordance was 10.8% to 15.1% with full plus partial concordance of 13.8% to 19.3%. Concordant mutations were associated with significantly higher variant allele frequency. Over half of mutations detected in either technique were not detected using the other biopsy technique. Including variants of unknown significance, the average number of alterations per patient was significantly higher for tissue (4.56) compared with ctDNA (2.16). When eliminating alterations not detectable in the ctDNA assay, mean number of alterations for tissue and ctDNA was similar (2.67 for tissue, 2.16 for ctDNA). Across five representative genes (TP53, PIK3CA, ERBB2, BRCA1, and BRCA2), sensitivity and specificity were 35.7% and 95.0%, respectively. Concordance when genomic alterations was detected in either tissue or ctDNA was low with each technique detecting a significant amount of nonoverlapping mutations. Potential explanations for the lack of concordance include tumor heterogeneity, different sequencing techniques, spatial and temporal factors, and potential germline DNA contamination. The study indicates that both tissue and blood-based NGS may be necessary to describe the complex biology of breast cancer. Mol Cancer Ther; 16(7); 1412–20. ©2017 AACR.

Background: Estrogen receptor (ER)-α is expressed in about 70% of breast cancers and drugs that target the receptor function, selective estrogen receptor modulators (SERM) and aromatase inhibitors (AIs) represent the standard of care for patients (pts) with ER+ breast cancer. Nevertheless, prolonged exposure to endocrine therapy may result in acquired resistance and subsequent progression of disease. Recent evidence showed that activating mutations (muts) in the ligand-binding domain of ER-α occur in approximately 20% of pts exposed to endocrine therapies and those genomic abnormalities may represent the driver of endocrine resistance. In this context, ctDNA provides a non-invasive source for real-time next generation sequencing (NGS) studies, in order to understand the biology of ABC and guide and monitor treatment. Methods: We conducted a retrospective review of 91 pts with ABC, including 57 pts with ER+ tumor, who had longitudinal assessment of their disease by ctDNA analysis. At the time of baseline sampling, 50/57 pts had stage IV cancer. The total number of blood samples collected was 184. 38 (67%) pts had serial samples. The average number of samples for each pt was 3 (range 1-7). The plasma-based assay was performed utilizing Guardant360 (Guardant Health, CA), a digital NGS technology to sequence a panel of > 50 cancer genes. Results: Among the ER+ subgroup (57 pts), we identified 11 pts (19%) harboring ESR1 muts in ctDNA. All 11 pts had metastatic disease: 2 (18%) had bone metastases, 2 (18%) had visceral metastases, 7 (64%) had both sites of disease. The median age was 55 years (range 33-73). 5 pts had inflammatory breast cancer. The most common ESR1 muts were: Y537S (6/11, 55%), D538G (4/11, 36%) and Y537N (3/11, 27%). 7 pts carried polyclonal muts. At the time of testing, 10 pts had already failed at least 1 line of endocrine therapy (average 2, range 1-5), including 6 pts that had received a fulvestrant-containing regimen, 8 pts ≥ 1 line of AIs. After the mut detection, 5 pt were on endocrine therapy and 4 pts were started on/continued chemotherapy. ESR1 muts disappeared in 2 pts (fulvestrant-palbociclib and chemotherapy respectively) who achieved stable disease as best response. Three pts continued to harbour muts and then progressed (one died). 2 pts had tissue NGS and ESR1 mut was not identified. Progression free survival and overall survival were 8 months (ms) and 21.5 ms in ESR1+ subpopulation versus 6.2 ms and 22.2 ms in the ESR1- pts (p = 0.78 and p = 0.97, respectively). At the time of analysis 5 pts were dead, 6 were currently alive. ESR1+ (n. pts) ESR1- (n. pts) Pts (total n.)1146 Previous chemotherapies11 (100%)31 (67%) Previous fulvestrant-containing regimens6 (54%)20 (43%) Previous AIs ± targeted therapy8 (73%)27 (59%) Conclusions: We observed that ESR1 muts, a known driver of endocrine resistance, occurs at a high frequency in heavily pre-treated estrogen receptor positive ABC. Blood-based diagnostics can be used to identify ESR1 muts sometimes not detected by tissue-based sequencing of the metastatic lesions indicating tumor heterogeneity and allowing dynamic monitoring of ABC. Citation Format: Rossi G, Lima Barros Costa R, Nagy RJ, Rademaker AW, Gradishar WJ, Santa-Maria CA, Curry-Edwards RL, Jain S, Flaum LE, Zagonel V, Platanias LC, Giles FJ, Talasaz A, Cristofanilli M. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA): A guide to the management of advanced breast cancer (ABC) [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 P1-05-06.

Purpose Liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), can be used to understand disease prognosis, tumor heterogeneity, and dynamic response to treatment in metastatic breast cancer (MBC). We explored a novel, 180-gene ctDNA panel and the association of this platform with CTCs and CTC clusters. Methods A total of 40 samples from 22 patients with MBC were included in the study. For the primary analysis, all patients had ctDNA sequencing using the PredicinePLUS™ platform. CTCs and CTC clusters were examined using the CellSearch™ System. Clinical and pathological variables were reported using descriptive analyses. Associations between CTC count and specific genomic alterations were tested using the Mann-Whitney U test. Results Of 43 sequenced patients, 40 (93%) had at least one detectable genomic alteration with a median of 6 (range 1–22). Fifty-seven different genes were altered, and the landscape of genomic alterations was representative of MBC, including the commonly encountered alterations TP53, PTEN, PIK3CA, ATM, BRCA1, CCND1, ESR1, and MYC. In patients with predominantly hormone-receptor-positive MBC, the number of CTCs was significantly associated with alterations in ESR1 (P GATA3 (P CDH1 (P CCND1 (P U test). Thirty-six percent of patients had CTC clusters, which were associated with alterations in CDH1, CCND1, and BRCA1 (all P U test). In an independent validation cohort, CTC enumeration confirmed significant associations with ESR1 and GATA3, while CTC clusters were significantly associated with CDH1. Conclusions We report on a novel ctDNA platform that detected genomic alterations in the vast majority of tested patients, further indicating potential clinical utility for capturing disease heterogeneity and for disease monitoring. Detection of CTCs and CTC clusters was associated with particular genomic profiles.

1084 Background: Metastatic breast cancer (MBC) is associated with genomic evolution, representing a challenge at clinical progression. While tissue and blood next-generation sequencing (NGS) allows for the baseline detection of alterations, non-invasive longitudinal assessment of ctDNA can provide a tool for monitoring tumor evolution. We characterized genomic changes using serial ctDNA testing in patients with clinical progression. Methods: Patient data was obtained under an IRB-approved protocol and ctDNA was collected at Northwestern University between 2015-2019. All ctDNA samples were analyzed using the Guardant360 NGS assay. Of 255 patients with MBC, 86 had at least two serial ctDNA collections with the second collection drawn at first progression (P1) by imaging and clinical assessment. Participants were followed until second clinical progression (P2). We analyzed type of alterations, mutant allele frequency (MAF), number of alterations (NOA), and sites of disease on imaging in close proximity to ctDNA evaluation. Matched pairs variations in MAF and NOA at P1 and P2 were tested through Wilcoxon test. Results: We identified 44 HR+, 20 HER2+ and 22 TNBC cases. Median lines of therapy were 3 (interquartile range (IQR): 1-6) for HR+, 3 (IQR: 1-5) for HER2+, and 2 (IQR: 1-4) for TNBC. The most likely alterations between baseline to P1 were TP53 (p < 0.0075), PIK3CA (p < 0.0126), AR (p < 0.0126), FGFR1 (p < 0.0455) and ESR1 (p < 0.0143). In the HR+ subset , ESR1 was statistically more likely at P1. ESR1 at P1 was also associated with development of new liver lesions (p < 0.0320). ERBB2 mutation at P1 was associated with new lung (p < 0.0050) or bone lesions (p < 0.0030). Increase in NOA was observed between baseline and P1 (p < 0.0001), P1 and P2 (p < 0.0001), and baseline to P2 (p < 0.0004). MAF was increased between baseline and P2 (p < 0.0480). Conclusions: Serial ctDNA testing identified resistance alterations ( TP53, PIK3CA, AR, ESR1, FGFR1), with some mutations indicating new sites of disease ( ESR1, ERBB2). Heterogeneity of ctDNA was significantly associated with progressive disease. Prospective evaluation of the impact of serial ctDNA testing on treatment decisions is needed to expand the role of precision medicine in MBC. [Table: see text]

Introduction: Advanced breast cancer can be more aggressive and portend a worse prognosis in the younger population (age < 50). While several studies have outlined clinico-pathologic features and molecular features of primary disease in young women with breast cancer, data in the metastatic setting remain scarce. Thus, the goal of this study was to explore the underlying clinic-pathologic and molecular profile of young women with metastatic breast cancer. Methods: We conducted a retrospective analysis of 138 females with metastatic breast cancer treated at Northwestern Medicine who provided consent for serial evaluation of circulating biomarkers. Patient were divided into two cohorts based on age at the time of metastasis, namely premenopausal (defined as age < 50) and postmenopausal (age ≥ 50). CellSearch™ immunomagnetic kit (Menarini Silicon Biosystems) was utilized to enumerate circulating tumor cells (CTCs), and the CellSearch™ CXC Kit (in 7.5 cc whole blood) characterized CTC HER2 expression. Circulating tumor DNA (ctDNA) was sequenced using the Guardant360 next-generation sequencing (NGS) assay (Guardant Health). When available, tissue samples from the primary and metastatic site(s) were sequenced using FoundationOne and/or Tempus xT NGS assays. Associations were drawn using Pearson’s χ2 test, independent samples T-tests, and multivariate logistic regression. Results: Of the 138 women, 54 (39%) were premenopausal with median age of 42 (range: 28-49), and 84 (61%) were postmenopausal with median age of 57 (range: 50-81). For the premenopausal cohort, 96% had invasive ductal carcinoma, 2% invasive lobular carcinoma and 2% mixed/unknown, compared to postmenopausal with 74%, 19% and 7% respectively, p=0.003. No statistically significant association was observed based on disease subtype (HR+/HER2-, HR+/HER2+, HR-/HER2+, TNBC), correspondingly stratified as 46%, 17%, 9%, 28% (premenopausal) and 57%, 10%, 13% 20% (postmenopausal). In total, 39% of the premenopausal and 40% of the postmenopausal patients had inflammatory breast cancer (IBC). Among patients initially diagnosed with non-metastatic disease, time (years) to metastasis was 2.76 (95% CI 2.11 to 3.41) for the premenopausal and 6.08 (95% CI 4.89 to 7.27) for the postmenopausal cohort, p=0.0001. Statistically significant associations were found when comparing the NGS datasets, derived from serial collection of ctDNA +/- tissue samples. Specifically, the premenopausal group had a higher incidence of GATA3 (11 vs 6 cases; p=0.017) alterations, but a lower incidence of NF1 (1 vs 18; p Conclusion: Our data reveal that premenopausal women diagnosed with metastatic breast cancer had a more rapid progression to metastasis and differ from their postmenopausal counterparts in both their pathologic profile (almost exclusively invasive ductal carcinoma) and molecular profile (notably, gene alteration frequencies of NF1, RB1 and GATA3), which could have significant implications in developing targeted treatment paradigms for younger women. Additionally, CTC prevalence in the metastatic setting differs from earlier stage breast cancer data showing a higher proportion of ≥5 CTCs in postmenopausal patients. Citation Format: Kristen Carroll, Ami M Shah, Lorenzo Gerratana, Chenyu Lin, Andrew A Davis, Qiang Zhang, Youbin Zhang, Lisa Flaum, Amir Behdad, Leonidas C Platanias, William J Gradishar, Massimo Cristofanilli. Clinico-pathological and molecular features in young women with metastatic 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 PD8-03.

Background: The clinical utility of routine clinical assessment of ctDNA in metastatic breast cancer (MBC) has not been clearly established. Recently, the Food and Drug Administration approved alpelisib (Piqray®) with a ctDNA-based companion diagnostic. Therefore, there is significant interest in describing the landscape of ctDNA alterations and establishing further clinical utility of this assessment. Methods: We retrospectively identified ctDNA data from a single institution (Northwestern University). All patients had next-generation sequencing performed by Guardant Health (Redwood City, CA) from 2015-2019 for clinical evaluation, and patients were consented for participation in a prospective registry study. Reports were evaluated for type of genomic alterations, number of alterations, and mutant allele frequency (MAF) of the dominant clone. Clinical information including demographics, pathology, imaging, treatment, and response data were collected via patient chart review. Associations were tested through Fisher’s exact test or Kruskal-Wallis test as statistically appropriate. Matched paired samples were investigated through McNemar test. Results: The cohort consisted of 255 patients with MBC. In total, 371 ctDNA reports were reviewed, including 116 patients with serial assessments. Baseline ctDNA evaluation occurred at a median of 3 lines of therapy (interquartile range (IQR): 1 - 5). Median number of alterations was 4 (IQR: 2-7), while median MAF was 4% (IQR: 0.6% - 17.9%). Across the entire cohort, PIK3CA and TP53 were the most commonly detected mutations, while MYC, FGFR1, PIK3CA, and ERBB2 were the most commonly detected amplifications. 40% of patients had PIK3CA aberrations observed in the analyzed plasma samples (28% amplification, 22% H1047R, 11% E542K), which was independent of breast cancer subtype. In the subset of patients with hormone-receptor positive (HR+) breast cancer, 30% of patients had ESR1 mutations (27% D538G, 19% Y537S, 12% Y537N, 11% E380Q). Patients with triple negative breast cancer (TNBC) had statistically higher MAF compared to HR+ or HER2+ breast cancer (P Conclusion: Liquid biopsy evaluation using ctDNA identified clinically meaningful genomic alterations at high frequency. In addition, our data indicate the potential clinical utility of ctDNA as a surrogate for tumor burden on imaging based on the observed correlation with lines of therapy received and sites of disease. Serial assessment of ctDNA demonstrated clonal evolution, particularly in the HR+ cohort, and identified resistant clones. Validation with an independent cohort is warranted. Citation Format: Andrew A Davis, Saya Jacob, Lorenzo Gerratana, Ami N Shah, Firas Wehbe, Neelima Katam, Qiang Zhang, Lisa Flaum, Kalliopi Siziopikou, Leonidas C Platanias, William J Gradishar, Amir Behdad, Massimo Cristofanilli. Landscape of circulating tumor DNA (ctDNA) in metastatic 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 P5-01-08.

BackgroundResponse rates to single agent immune checkpoint blockade in unselected pretreated HER2−negative metastatic breast cancer (MBC) are low. However, they may be augmented when combined with chemotherapy.MethodsWe conducted a single-arm, phase II study of patients with triple negative (TN) or hormone receptor-positive endocrine-refractory (HR+) MBC who were candidates for capecitabine. Patients were treated with pembrolizumab 200 mg intravenously day 1 and capecitabine 1000 mg/m2by mouth twice daily on days 1–14 of a 21-day cycle. The primary end point was median progression-free survival (mPFS) compared with historic controls and secondary end points were overall response rate (ORR), safety and tolerability. The study had 80% power to detect a 2-month improvement in mPFS with the addition of pembrolizumab over historic controls treated with capecitabine alone.ResultsThirty patients, 16 TN and 14 HR+ MBC, were enrolled from 2017 to 2018. Patients had a median age of 51 years and received a median of 1 (range 0–6) prior lines of therapy for MBC. Of 29 evaluable patients, the mPFS was 4.0 (95% CI 2.0 to 6.4) months and was not significantly longer than historic controls of 3 months. The median overall survival was 15.4 (95% CI 8.2 to 20.3) months. The ORR was 14% (n=4), stable disease (SD) was 41% (n=12) and clinical benefit rate (CBR=partial response+SD>6 months) was 28% (n=8). The ORR and CBR were not significantly different between disease subtypes (ORR 13% and 14%, CBR 25% and 29% for TN and HR+, respectively). The 1-year PFS rate was 20.7% and three patients have ongoing responses. The most common adverse events were low grade and consistent with those seen in MBC patients receiving capecitabine, including hand-foot syndrome, gastrointestinal symptoms, fatigue and cytopenias. Toxicities at least possibly from pembrolizumab included grade 3 or 4 liver test abnormalities (7%), rash (7%) and diarrhea (3%), as well as grade 5 hepatic failure in a patient with liver metastases.ConclusionsCompared with historical controls, pembrolizumab with capecitabine did not improve PFS in this biomarker unselected, pretreated cohort. However, some patients had prolonged disease control.Trial registration numberNCT03044730.

Determining the need for adjuvant chemotherapy in estrogen receptor (ER)+ disease can be influenced by pathological characteristics and gene expression assays [i.e., Oncotype Dx recurrence scores (RSs)]. The primary objective of this study is to investigate the relationship between the RSs and pathological markers in younger ( 50) versus older (≥ 50) women with early-stage node-negative ER+ breast cancer.This was a single academic-center retrospective cohort study. Subjects who underwent Oncotype gene expression testing were retrospectively and sequentially identified. 436 Subjects were identified of which 344 were eligible for analysis (133 younger subjects 50 years of age, and 211 older subjects ≥ 50 years). Pathological data assessed included the progesterone receptor (PR), histological grade (grade), Ki-67, and P53. A multivariable regression analysis was performed using age, PR, and grade as predictor variables for RS. Adjusted RBased on the multivariable regression analysis, the adjusted RThe relationship between pathological features and RS is consistent irrespective of age; therefore, models predicting RS may be applicable irrespective of age.

Immune checkpoint inhibitors produce modest responses in metastatic breast cancer, however, combination approaches may improve responses. A single arm pilot study was designed to determine the overall response rate (ORR) of durvalumab and tremelimumab, and evaluate immunogenomic dynamics in metastatic endocrine receptor (ER) positive or triple negative breast cancer (TNBC). Simon two-stage design indicated at least four responses from the first 18 patients were needed to proceed with the second stage. T-cell receptor (TCR) sequencing and immune-gene expression profiling were conducted at baseline and two months, whole exome sequencing was conducted at baseline. Eighteen evaluable patients were accrued (11 ER-positive; seven TNBC). Only three patients had a response (ORR = 17%), thus the study did not proceed to the second stage. Responses were only observed in patients with TNBC (ORR = 43%). Responders versus non-responders had upregulation of CD8, granzyme A, and perforin 1 gene expression, and higher mutational and neoantigen burden. Patients with TNBC had an oligoclonal shift of the most abundant TCR-beta clonotypes compared to those with ER-positive disease, p = 0.004. We conclude responses are low in unselected metastatic breast cancer, however, higher rates of clinical benefit were observed in TNBC. Immunogenomic dynamics may help identify phenotypes most likely to respond to immunotherapy.

A majority of breast cancers are hormone receptor (HR) positive and are responsive to various types of hormone manipulation. Endocrine therapy is the preferred first-line therapy for patients with advanced estrogen receptor (ER) positive, HER2-negative breast cancer who do not have symptomatic visceral disease. Endocrine therapy is often continued in the second- and third-line setting, with chemotherapy deferred until tumor becomes endocrine therapy refractory and/or a visceral crisis in imminent. Therapeutic options vary based on clinical presentation and include single-agent therapies such as tamoxifen, aromatase inhibitors and fulvestrant, and combination therapies options. Over the past few years, multiple trials have shown significant improvement in outcomes when endocrine therapy is combined with CDK 4/6 inhibitors or mTOR inhibitors. Improved efficacy comes at a cost of a modest increase in toxicity. Mechanisms of ER resistance have been defined leading to multiple strategies to improve efficacy and overcome resistance. These include the combination therapies options mentioned above and other novel drugs that are in development. This review will summarize the existing literature regarding endocrine therapy in postmenopausal metastatic breast cancer and outline treatment approaches in the first-line metastatic setting and beyond.

The prolactin (PRL) receptor is over-expressed in breast cancer, and pre-clinical data indicate that it contributes to breast oncogenesis. Cabergoline is a potent dopamine receptor agonist of D2 receptors and has a direct inhibitory effect on pituitary PRL secretion. A phase II study of cabergoline in patients with metastatic breast cancer was conducted. The primary end point of the study was to determine the clinical benefit rate (CBR) at 2 months. Eligible patients had tumors of any receptor status with no limit of prior lines of therapy. Measurable and unmeasurable diseases were allowed. Cabergoline 1 mg orally, twice weekly (1 cycle = 4 weeks) was given until disease progression or unacceptable toxicity. PRL receptor immunohistochemical staining was performed on available baseline tumor tissue; serial serum PRL levels were assessed. Twenty women were enrolled; 18 were evaluable for CBR. Tumor receptor status was distributed as follows: HR−any/HER2+ 2(10%), HR+/HER2− 18 (90%). The CBR was 33% (6/18), median progression free survival was 1.8 months, and median overall survival was 10.4 months. Two patients experienced disease control for over 12 months. Most common treatment-related adverse events were nausea (30%), fatigue (25%), and elevation in alkaline phosphatase (15%). Nine patients had baseline tissue for analysis; there was no association between baseline tumor PRL receptor expression and clinical benefit (p = 0.24). Change in serum PRL level and response were not correlated after 2 months of treatment (p = 0.64). Cabergoline was well tolerated, and while the ORR was low, a small subset of patients experienced extended disease control.

Introduction: CTCs play a critical role in MBC, and a portion of CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of forming distant metastasis in MBC as compared to individual CTCs. However, genomic characterization of CTCs-clusters remain largely because the enrichment of CTC-clusters is technically challenging. Herein, we describe a novel isolation workflow to select CTC-clusters expressing specific biomarkers for patients with MBC. Methods: Whole blood samples (7.5ml/each) were collected from 5 patients with stage III/IV BCa patients. CTC enumeration was performed with the FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs, and immunofluorescent staining reagents including Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocyte), and anti-HER-2/neu-FLU. Or, using CXC kit includes anti-CCR5-PE. After confirming CTCs-clusters, both CTCs and CTC-clusters were enriched from Celltracks cartridge and were loaded into DEPArrayTM NxT cartridge, and then were isolated and sorted by using fluorescent imaging based DEPArrayTM NxT System. Results: All these samples were identified having CTCs (≥ 5, between 34 to 208 CTCs) with CTC-clusters (between 1 to 16) by CellSearch analysis. The images of CTCs and CTC-clusters were displayed in CellBrowserTM by the DEPArrayTM NxT System. According to multiple channels assigned for PE, DAPI, APC and FITC, the CTCs and CTC-clusters were classified and then sorted based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-, with additional markers of HER2 or CCR5. The targeted CTCs and CTC-clusters were routed, parked and then recovered under the direction of Recovery ManagerTM. By using this technique, we successfully recovered and harvested 27/34 (DEPArray harvest/CellSearch diagnosis), 54/151, 57/115, 75/196, and 115/208 CTCs from each CellSearch cartridge sample respectively. Meanwhile, we collected 1/1 (100%), 6/16 (37.5%), 1/1 (100%), 3/11 (27.3%), and 4/4 (100%) of CTC-clusters respectively, as each CTC-cluster includes average of 2.85 CTCs. CTC-clusters at diameter as much as 45µm could be routed and recovered smoothly. Furthermore, CTC-Clusters with biomarkers of interest such as HER2+ or CCR5+ expression were separated into different tubes. The white blood cells from the same samples were also selected successfully as the controls. Conclusions: We first reported a new workflow for CTC-clusters isolation. With further optimization, this feasible and reliable strategy will help streamline isolation of single CTC and CTC-clusters in MBC patients for genomic analysis providing the opportunity to gain new insights on the molecular mechanisms associated with the metastasis process. Citation Format: Qiang Zhang, Lorenzo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Amir Behdad, Firas Wehbe, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. A novel application of DEPArrayTM NxT System to isolate circulating tumor cell (CTC)-clusters from patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1351.

Introduction: The Chemokine C-C motif ligand 5 (CCL5) and its receptor 5 (CCR5) play a significant role in solid tumors, particularly triple negative breast cancer (TNBC) and HER2+ subtypes with prognostic implications. CCL5-CCR5 axis was reported to govern cancer stem cells expansion and play key role in MBC progression. Our group recently reported that overexpression of HER2 was associated with CTC-clusters which caused poor prognosis of patients with MBC. Herein, we first reported the expression of CCR5 in CTCs of MBC patients, and described the correlation between CCR5 and HER2 expression in CTCs. Methods: Whole blood samples (7.5ml/each) were collected from stage III/IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, and immunofluorescent staining reagents including anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. After confirming CTCs were positive by CellSearch system, CCR5 expression were evaluated by using CXC kit for multiple staining includes anti-CCR5-PE (R&D Systems), Anti-CK-FITC, DAPI and Anti-CD45-APC. The correlation between HER2 expression and CCR5 expression was analyzed by Kruskal-Wallis test was used for statistics. Results: CTCs were found positive (≥5) in all seven MBC patients with a range of numbers between 124 and 442, and HER expression was identified in 6 out 7 cases (between 57 and 149 total cells). The ratios of HER2+ CTCs/total CTCs were varied between 28.5% and 88.1%. Meanwhile, CCR5 expression were found in 3 out 7 patients (Group 1), with CCR5+ CTCs/ total CTCs ratios were 4.45%, 43.1% and 59.1% respectively. There were 4 patients without CCR5 expression in CTCs (Group 2). The average HER2+ CTCs was 127.6 with HER2+ CTC/Total CTC as average of 59.4% in Group 1, which were significantly higher in compared with the corresponding numbers as 80.3 and 29.97% respectively in Group 2, which indicated that upregulation of CCR5 was positively associated with high level of HER2 expression in CTCs. Conclusions: Our data provides the first evidence of strong expression of CCR5 in CTCs of MBC as potential new marker. The significant correlation between overexpression of HER2 in CTCs and high level of CCR5 indicated that CCR5 may contributes to more aggressive MBC subtypes with HER2 expression, which promotes carcinogenesis and metastasis at least partly by maintaining and increasing cancer stem cells leading to increased invasion. We conclude that the further understanding of the molecular interactions of CCR5 and HER2 in CTCs will be important to elucidate the mechanism of metastasis of MBC and predict prognosis. Citation Format: Qiang Zhang, Lorezo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Richard G. Pestell, Firas Wehbe, Amir Behdad, Leonidas Platanias, William Gradishar, Massimo Cristofanilli. Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 408.

Introduction: Molecular and genomic characterization of CTCs may help to understand MBC prognosis and predict treatment benefit. We previously reported that overexpression of HER2 in CTCs is associated with detection of clusters, more aggressive clinical features and prognosis in MBC (2018 AACR #5195). Herein, we report a new finding that a widespread variation on chromatin distribution in CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV BCa patients before therapy. CTC enumeration were performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs. After confirming CTCs were positive by CellSearch system, live CTCs enrichment was performed by using Parsortix system (ANGLE) utilizes microfluidic based 10μm separation cassettes. The captured CTCs were stained by Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER2-FITC. CTCs chromatin packing density was scanned by Partial Wave Spectroscopic (PWS) microscopy which is a label-free spectroscopic microscopy method that resolves structures in cells and quantify the cell nucleus heterogeneity of chromatin packing density scaling between 20-350 nm, or from the kb-pair to 10 Mb-pair range. For each nuclei, the nanoscale heterogeneity of chromatin packing was analyzed as nuclear statistical parameter standard deviation Σ (sigma, RMS). Kruskal-Wallis test was used for statistics. Results: We identified 400 CTCs by CellSearch system, including 115 HER2+ CTCs and 285 HER2- CTCs. The live CTCs were sorted by Parsortix system and were proceeded with multiple staining and scanning by PWS microscopy. CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. There were 97 acquired nuclei that were scanned and evaluated successfully, including 17 HER2+ CTCs, 12 HER2- CTCs and 80 non-CTC cells. According to the images, stronger nanoscopic variations at each pixel in internal structure represents higher chromatin distribution heterogeneity quantified by RMS. The average RMS was 0.05125 in CTC cells which is significantly higher than the non-CTC population (RMS=0.0381, p Conclusion: This is the first report of chromatin heterogeneity in CTCs, and the association between HER2 expression and high variations of chromatin distribution in MBC patients. This novel property of CTCs describing nanoscale heterogeneity of chromatin packing requires further evaluation and validation but, it may offer a novel dimension in the understanding of the metastatic process. Citation Format: Qiang Zhang, Lorenzo Gerratana, Ami N. Shah, Andrew Adam Davis, Lisa Flaum, Xiang Zhou, Youbin Zhang, Vadim Backman, Amir Behdad, Firas Wehbe, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased chromatin heterogeneity in circulating tumor cells (CTCs) is associated with high levels of HER2 expression in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 429.

1096 Background: Response rates to single agent immune checkpoint blockade in unselected MBC are low; however, they may be augmented when combined with chemotherapy. Methods: We conducted a single-arm, phase II study of patients with TN or endocrine-refractory MBC who were candidates for capecitabine. Patients were treated with pembrolizumab 200 mg IV day 1 and capecitabine 1000 mg PO BID days 1-14 of a 21-day cycle. The primary endpoint was progression free survival (PFS) and secondary endpoints were overall response rate (ORR), safety and tolerability. The study had 80% power to detect a 2 month (mo) improvement in mPFS with the addition of pembrolizumab over historic controls treated with capecitabine alone. Results: Thirty patients, 16 with TN and 14 endocrine-refractory MBC, were enrolled from 2017-18. Patients had a median age of 51 years and received a median of 1 prior line of therapy for MBC. Of 29 evaluable patients, the mPFS was 4.1 mo (95% CI, 2.3-8.2 mo) and median overall survival was 15.4 mo (95% CI, 8.2-16.6 mo). ORR was 14% (n = 4), stable disease (SD) was 41% (n = 12), and clinical benefit rate (CBR = PR + SD > 6 mo) was 28% (n = 8). The ORR and CBR were similar between disease subtypes (ORR 13% and 14%, CBR 25% and 29% for TN and endocrine refractory, respectively). The 1-year PFS rate was 20.7% and 3 pts have ongoing responses. The most common adverse events were low grade and consistent with those seen in MBC patients receiving capecitabine, including hand-foot syndrome, gastrointestinal symptoms, fatigue, and cytopenias. Toxicities at least possibly from pembrolizumab included grade 3 or 4 liver test abnormalities (7%), rash (7%), and diarrhea (3%), as well as grade 5 hepatic failure in a pt with liver metastases. Conclusions: Compared to historical controls, pembrolizumab with capecitabine did not improve PFS in this biomarker unselected cohort; however, some patients had prolonged disease control. Future studies of chemo-immunotherapy in MBC with liver metastases require close safety monitoring. Clinical trial information: NCT03044730.

3038 Background: Liquid biopsy provides real-time data about prognosis and actionable mutations in MBC. The aim of this study was to explore the combination of ctDNA analysis and CTCs enumeration in estimating target organs more susceptible to MBC involvement. Methods: This retrospective study analyzed 85 MBC patients (pts) characterized for both CTCs and ctDNA at baseline. CTCs were isolated through the CellSearch kit (Menarini Silicon Biosystems, PA), while ctDNA was analyzed using the Guardant360 NGS-based assay (Guardant Health, CA). Pts with ≥ 5 CTC/7.5 ml of blood were defined as Stage IV aggressive as previously reported (Cristofanilli et al 2019). Statistical associations were explored through uni- and multivariate logistic regression and Fisher’s exact test. Results: 37% of pts were diagnosed with hormone receptor positive (HRpos) MBC, 26% with HER2-positive MBC and 37% with triple negative MBC (TNBC), 28 pts (33%) were defined as stage IV aggressive. The most observed metastatic sites were bone (37%), lymph nodes (29%), lung (27%) and liver (25%). In multivariate analysis, IBC and ESR1 mutations were the only significant factors associated with liver metastases (respectively, OR 0.12, P = 0.038 and OR 24.01, P = 0.019), while no associations were found with respect to lung localizations. Intriguingly, all HRpos MBC pts with ESR1 mutations had bone metastases (P = 0.022), while IBC and Stage IV aggressive were independently associated with bone metastases (respectively OR 0.10, P = 0.006 and OR 19.92, P = 0.003). FGFR1 and NF1 were associated with lymph node localizations (OR 3.68, P = 0.046, OR 4.39, P = 0.031, respectively), while CDK6 and TP53 alterations were associated with serosal involvement (OR 14.34, P = 0.029, OR 0.08, P = 0.031, respectively). Notably, TNBC and IBC were both associated with soft tissue spreading (respectively OR 3.7, P = 0.011, OR 2.79, P = 0.018). Conclusions: These results suggest that ctDNA and CTCs enumeration could give useful insights on MBC organotropism, suggesting a possible role for future monitoring strategies that dynamically focus on high-risk organs defined by tumor biology.

1036 Background: The monitoring of CTCs and cfDNA in metastatic BCa showed ability to predict treatment resistance and survival. Here we report a highly significant correlation between HER2 alterations and ESR1 mutations of cfDNA with CTCs and prognosis of BCa, which may help to predict disease recurrence and treatment benefit. Methods: A total of 85 blood samples (7.5ml/each) were collected from 85 patients with stage III/IV BCa who received treatments at Northwestern RHLCCC. CTC enumeration was performed in FDA approved CELLTRACKS ANALYZERII System (Menarini). Plasma cfDNA was analyzed using Guardant360 NGS-based assay including a 73-gene panel for genomic alterations or mutations. We previously reported cut-off of 5.7% (2018 ASCO) was used to dichotomize the prognostic value of cfDNA percentage. Kruskal-Wallis test was used for statistics. Results: Of the 85 whole samples analyzed, there were 72 samples and 67 samples without ESR1 mutations (ESR1-) and HER2 alterations ( HER2-) respectively, and there are 13 samples and 18 samples that had ESR1 mutations ( ESR1+) and HER2 alterations ( HER2+, 10 amplified, 7 mutated, 1 for both) respectively. CTC positive (≥5) were detected in 13/57 ESR1-HER2- samples (Group 1) and 5/15 ESR1- HER2+ samples (Group 2), 7 /10 ESR1+ HER2- samples (Group 3), 3/3 ESR1+ HER2+ samples (Group 4). The median CTCs number/sample in Group 3 (15 CTCs) and Group 4 (12 CTCs) were significantly higher than Group 1 (0 CTC) and Group 2 (2 CTCs) (P = 0.0020). There were a significant higher average metastasis sites in Group 3 (3 sites) and Group 4 (3 sites) in compared to Group 1 (2 sites) and Group 2 (1 site) (P = 0.0035). Furthermore, patients in Group 4 ( ESR1+ HER2+) has the worst prognosis in compared to other groups (P = 0.0151) on overall survival. Conclusions: Both ESR1 mutations and HER2 alterations in cfDNA contribute to CTCs detection and disease metastasis sites independently, when ESR1 mutations plays a major role. The synergy of ESR1 mutation and HER2 alteration expands the predictive role of liquid biopsy tests monitoring the metastatic prognosis and endocrine resistance for clinical decision-making.

Background: Molecular diagnostic, in particular next-generation sequencing (NGS) technologies, improved the detection of actionable mutations (muts) in MBC at baseline and recurrence. We evaluated the ability of ctDNA to detect molecular abnormalities, monitor disease progression and predict outcome. Methods: We conducted a retrospective study of 91 patients (pts) with locally advanced and MBC, who had longitudinal assessment of their disease by ctDNA analysis. The plasma-based assay was performed utilizing Guardant360 (Guardant Health, CA), a digital NGS technology to sequence a panel of > 50 cancer genes. After tabulating number of muts and quantification of overall ctDNA detected for every patient at baseline, a receiver operating characteristic (ROC) analysis was performed to identify the best cut-offs that separated the pts who had a disease progression from those who hadn't, and the patients who died from those still alive. The overall survival (OS) analysis has been performed using Kaplan-Meier curves. Results: 84 pts (92%) had stage IV cancer. 63% cases were ER+, 27% HER2+, 29% TNBC. 277 blood samples were collected and 84% had muts. 65% of the pts had serial samples. The average number of alterations detected in each sample was 3 (0-27) and the average ctDNA fraction detected was 4.5% (0-88.2%). The most common alterations were: TP53 (52%), PIK3CA (40%), ERBB2 (20%), NOTCH1 (15.5%), APC (14%), MET (13%). 16 pts (19%) were initiated on a targeted therapy based on ctDNA test results. At the time of analysis 36 pts (39.6%) were dead, 55 (60.4%) were currently alive. PFS was 5.2 months (ms) and OS was 21.5 ms. A statistically significant difference in PFS and OS by log rank test was found between % ctDNA at baseline < 0.5 versus ≥ 0.5 (p = 0.003 and p = 0.012, respectively) and number of muts at baseline < 2 versus ≥ 2 (p = 0.059 borderline and p = 0.0015). Moreover, a statistically significant association by Fisher's exact test was found between the number of alterations and the % ctDNA detected in the baseline sample (% of pts with muts ≥ 2 was 19% when % ctDNA < 0.5%, versus 85% when % ctDNA ≥ 0.5%; p < 0.0001). PFS (ms) p = 0.059 (log rank test)Muts < 2 (n = 32)Muts ≥ 2 (n = 58)658%40%1230%13%1821%6%24--PFS(ms) p = 0.003 (log rank test)% ctDNA < 0.5(n = 27)% ctDNA ≥ 0.5(n = 60)665%39%1241%10%1823%6%24-- OS(ms) p = 0.002 (log rank test)Muts < 2(n = 32)Muts ≥ 2(n = 57)697%66%1288%51%1888%42%24-29%OS(ms) p = 0.012 (log rank test)% ctDNA < 0.5(n = 27)% ctDNA ≥ 0.5(n = 59)696%69%1290%55%1885%48%24-35% Conclusions: ctDNA liquid biopsy provides a real-time, quantitative NGS-based assessment of MBC which is useful for treatment planning, disease monitoring and prognostic evaluation. Future prospective studies should consider the use of ctDNA for molecular and prognostic stratification. Citation Format: Rossi G, Austin LK, Nagy RJ, Rademaker AW, Gradishar WJ, Santa-Maria CA, Curry-Edwards RL, Jain S, Flaum LE, Lima Barros Costa R, Zagonel V, Platanias LC, Giles FJ, Talasaz A, Cristofanilli M. Circulating tumor DNA (ctDNA): A real-time application of precision medicine to the management of metastatic breast cancer (MBC) [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 PD1-02.

Background: Liquid biopsy technologies, including CTCs and ctDNA, may provide prognostic information for advanced breast cancer (ABC) patients. We evaluated quantification and molecular characterization of ctDNA-based testing for prognostic stratification and predictive analysis. Methods: This retrospective study analyzed a cohort of 91 patients (pts) with ABC, which was longitudinally characterized for CTCs and ctDNA. CTCs were isolated through the CellSearch immunomagnetic kit (Menarini Silicon Biosystems, PA), and ctDNA was analyzed using the Guardant360 NGS-based assay (Guardant Health, CA). A receiver operating characteristic (ROC) analysis was performed to identify the best ctDNA percentage (%ctDNA) cut-off capable to separate pts with ≥ 5 CTC/7.5 ml of blood. Survival analyses for both CTC and %ctDNA were investigated by log-rank test. Associations between %ctDNA stratification and detectable ctDNA mutations were explored through Fisher's exact test, and multiplicity was corrected through Bonferroni-Holm method (Q value). Results: 40 pts (44%) were diagnosed with luminal-like ABC, 25 pts, (27%) with HER2-positive ABC, and 26 pts (29%) with triple negative ABC. 84 pts (92%) had stage IV disease and 26 pts (31%) were de novo metastatic. 22 pts (35%) had a CTC count ≥ 5, while among pts with CTC < 5, 21 (34%) had no detectable CTCs. The median %ctDNA was 1.65 (IQR: 0.3 - 9.8), and 11 pts had no detectable ctDNA. A cut-off of 5.7 was selected (AUC: 0.7533) by ROC analysis. A significant association for both PFS and OS was observed for the selected threshold (log-rank test P=0.024 and P=0.0015, respectively). Pts with a %ctDNA < 5.7 had a median PFS and OS of 6.2 ms (IQR: 2.1 - 12.1) and 21.7 ms (IQR: 12.82 - not reached), respectively. For pts with a %ctDNA ≥ 5.7, median PFS and OS were 3 ms (IQR: 2.1 - 7) and 7.2 ms (IQR: 7.2 - not reached), respectively. The prognostic impact of CTCs was confirmed for PFS and OS (P=0.024 and P Conclusions: The CTCs-derived %ctDNA cut-off is capable of prognostic stratification in ABC. Interestingly, ctDNA can stratify pts also without detectable CTCs, providing valuable information on prognosis, mutational tumor burden and disease monitoring and characterization. Further prospective studies are needed to validate these findings. Citation Format: Lorenzo Gerratana, Andrew A. Davis, Giovanna Rossi, Kimberly Strickland, Zhaomei Mu, Lisa Flaum, William J. Gradishar, Cesar Santa-Maria, Hushan Yang, Massimo Cristofanilli. The liquid biopsy network in advanced breast cancer (ABC): Benchmarking the prognostic role of circulating tumor DNA (ctDNA) to the goal standard circulating tumor cells (CTCs) enumeration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2646.

Introduction: Molecular and genomic characterization of CTCs may help to understand breast cancer prognosis and predict treatment benefit, especially for metastatic or recurrent disease. Overexpression of HER2 protein has been associated with rapid cell division and the metastasis and prognosis of advanced breast cancer in the clinic. Herein, we report a correlation between the detection of CTC clusters and HER2 overexpression in otherwise biomarker negative disease. Methods: 146 whole blood samples (7.5ml/each) were collected from 95 patients with stage III/IV breast cancer at baseline, before and after systemic therapy. All samples were transported and stored at 15-30 °C in EDTA tubes. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search) contains ferrofluid-based capture reagent particles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs. Furthermore, it includes immunofluorescent staining reagents including Anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-. Database of CTCs was generated and linked with clinical database. Kruskal-Wallis test was used for statistics. Results: Of the 146 samples analyzed, 80 samples contained CTCs, among which 54 cases did not form clusters (Group 1), 11 samples had 1-5 clusters (Group 2), and 15 had >5 clusters (Group 3). The average ratio of CTCs/ total EpCAM+ was directly correlated with the number of clusters and were reported as 15.36%, 25.32% and 43.65% for the 3 cohorts respectively (P Conclusion: We demonstrate that patients with detectable CTCs clusters in comparison with CTCs with no or low numbers of clusters, high levels of CTCs clusters (> 5 clusters/7.5ml blood) is a strong indicator for more aggressive of CTCs with high level of HER2 expression, which tend to be more aggressive in metastasis and worse prognosis than HER2-negative breast cancers. Along with and cancer stage and HER2 status, CTCs clusters helps determine your treatment options in clinic. Citation Format: Qiang Zhang, Youbin Zhang, Lisa Flaum, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5195.

Introduction: Breast cancers (BCa) in younger women are more likely to be fast-growing, higher grade and hormone receptor-negative. As such these tumors tend to be associated with a worse prognosis even after systemic treatments with surgery, chemotherapy and radiation. The mechanisms that contribute to these tumors' aggressiveness remain largely unknown. Surveillance of circulating tumor cells (CTCs) may help to understand BCa prognosis and predict treatment benefit, especially for women with metastatic disease. Herein, we report a new finding of the correlation between age and the changes of CTC after the systemic therapies. Methods: 160 whole blood samples (7.5ml/each) were collected from 100 patients with stage III/IV BCa at baseline, before and after systemic therapy. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search). CTCs were confirmed using Anti-CK-PE, CD25, and EpCaM antibody and DAPI nuclear stain. Anti-CD45-APC antibodies were used to identify leukocytes. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD25-. Database of CTCs was generated and linked with clinical database. Student test was used for statistics. Results: Among 100 subjects who met inclusion criteria, 54 subjects (68%) at least 1 detectable CTCs in the baseline blood drawn prior to treatment. Of those, 37 patients had ≥ 5 CTCs, 17 patients had 1-5 CTCs, and 46 patients had no CTCs. There was no significant difference on the average age between these CTCs groups, which are 54.89, 53.52 and 53.30 respectively (P>0.05). Among the initial cohort, 42 subjects had at least 2 blood draws before and after systemic therapies respectively. Among these 42 patients, 11 had more CTCs after systemic therapy [Group 1 (26%), the increased range of CTC is from 1 to 756] and 17 patients had no CTCs before or after therapy [Group 2 (40%)]. There were 14 patients who had significantly fewer CTCs after systemic therapy [Group 3 (34%), the decreased range of CTC is from -1 to -23]. There was a significant difference in the average ages of each group (48.45, 56.00 and 57.28 years respectively; P=0.02). The results indicated that younger age (less than 50 years old) is associated with persistent of higher CTCs after systemic therapy likely reflecting decreased benefit from systemic therapy. Conclusion: Our original findings suggest age-related differences in CTCs response after systemic therapies for advanced BCa. These findings may indicate that younger patients may be at higher risk of developing more distant metastases as suggested by the increased numbers of CTCs after the systemic therapies. We propose that a combination of baseline CTCs detection and age be considered as a potential new criteria for selecting the systemic therapies for BCa patients. Citation Format: Qiang Zhang, Lorenzo Gerratana, Lisa Flaum, Youbin Zhang, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) after systemic therapy is associated with younger age in stage III/IV breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1597.

12040Background: Liquid biopsy technologies, including CTCs and ctDNA, have a growingly paramount role in the management of advanced breast cancer (ABC) and are potentially capable to convey differ...