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PURPOSE Given the heterogeneity and improvement in outcomes for metastatic breast cancer (MBC), we developed a staging system that refines prognostic estimates for patients with metastatic cancer at the time of initial diagnosis, de novo MBC (dnMBC), on the basis of survival outcomes and disease-related variables. METHODS Patients with dnMBC (2010-2016) were selected from the National Cancer Database (NCDB). Recursive partitioning analysis (RPA) was used to group patients with similar overall survival (OS) on the basis of clinical T category, grade, estrogen receptor (ER), progesterone receptor, human epidermal growth factor receptor 2, histology, organ system site of metastases (bone-only, brain-only, visceral), and number of organ systems involved. Three-year OS rates were used to assign a final stage: IVA: >70%, IVB: 50%-70%, IVC: 25 to RESULTS At a median follow-up of 52.9 months, the median OS of the original cohort (N = 42,467) was 35.4 months (95% CI, 34.8 to 35.9). RPA stratified patients into 53 groups with 3-year OS rates ranging from 73.5% to 5.7%; these groups were amalgamated into four stage groups: 3-year OS, A = 73.2%, B = 61.9%, C = 40.1%, and D = 17% (log-rank P < .001). After bootstrapping, the survival outcomes for the four stages remained significantly different (log-rank P < .001). This staging system was then validated using SEER data (N = 20,469) and a separate cohort from the NCDB (N = 7,645) (both log-rank P < .001). CONCLUSION Our findings regarding the heterogeneity in outcomes for patients with dnMBC could guide future revisions of the current American Joint Committee on Cancer staging guidelines for patients with newly diagnosed stage IV disease. Our findings should be independently confirmed.

PURPOSE Chemotherapy has not demonstrated benefit over adjuvant endocrine therapy alone for postmenopausal patients with node-positive breast cancer with a 21-gene breast recurrence score (RS) of 25 or below (RS ≤ 25). We tested whether combined results from RS and the sensitivity to endocrine therapy (SET2,3) index of endocrine-related transcription (SETER/PR) adjusted for baseline prognostic index (BPI) improve prognostic assessment, and whether SET2,3 predicted benefit from anthracycline-based chemotherapy. METHODS A blinded retrospective clinical validation of SET2,3 in two randomized treatment arms from the SWOG S8814 trial comparing adjuvant anthracycline-based chemotherapy followed by tamoxifen endocrine therapy for 5 years, versus tamoxifen alone. SET2,3 assay was calibrated and measured using whole-transcriptome RNA sequence of tumor samples already tested for RS. The primary end point was disease-free survival (DFS). RESULTS There were 106 events in 283 patients over a median follow-up of 8.99 years. Proportional hazards assumptions were met during the first 5 years only. SET2,3 index and RS were not correlated (r = –0.04) and were independently prognostic (SET2,3: hazard ratio [HR], 0.48 per unit; 95% CI, 0.34 to 0.68; P < .001; RS: HR, 1.28 per 10 units; 95% CI, 1.14 to 1.44; P < .001). SET2,3 index did not predict chemotherapy benefit (interaction P = .77). SET2,3 was high in 93/175 (53%) patients with RS ≤ 25 (concordant low-risk), with 5-year DFS 97%. SET2,3 was low in 55/108 (51%) patients with RS > 25 (concordant high-risk), with 5-year DFS 53%. Both components of SET2,3 index were prognostic after adjustment for RS: SETER/PR (HR, 0.65; 95% CI, 0.46 to 0.92) and BPI (HR, 0.45; 95% CI, 0.31 to 0.64). CONCLUSION SET2,3 index was not correlated with RS, demonstrated additive prognostic performance, and was not chemopredictive in this subset of patients from S8814. The SETER/PR and BPI components of SET2,3 each added prognostic information to RS.

Background: Late recurrence of breast cancer after 5 years accounts for about 50% of recurrences in hormone receptor (HR)-positive early breast cancer (EBC). TAILORx established non-inferiority of adjuvant endocrine therapy (ET) given for at least 5 years to chemotherapy plus ET (CET) in EBC and a 21-gene recurrence score (RS) of 11-25, although there was some chemotherapy benefit in women Methods: Eligibility criteria included women 18-75 years with HR-positive, HER2-negative, T1b-T2N0 EBC who agreed to have CT assigned or randomized based on the RS assay. The primary endpoint was invasive disease-free survival (iDFS) in the RS 11-25 group. The “primary analysis” refers to the original prespecified analysis for the primary IDFS endpoint (836 IDFS events at full information in the RS 11-25 group) after a median of 7.5 years. The “updated analysis” was performed after a median followup of 11.0 and 10.4 years in the randomized and overall populations, respectively. Results:10,253 eligible women enrolled between 4/7/06-10/6/10.The updated analysis includes substantially more events that the primary analysis, including IDFS events (1819 vs. 1210), distant recurrences (561 vs. 384), locoregional +/- distant recurrences (764 vs. 543), and deaths (910 vs. 499). The table provides 5 and 12-year event rates (and standard errors) for all arms, and comparisons of the randomized arms. The primary trial conclusions remain unchanged: ET was non-inferior to CET in the randomized group with a RS 11-25. Although recurrence occurred in < 10% by 12 years for a RS 0-25, late recurrence events beyond 5 years exceeded earlier recurrence. Non-recurrence events occurred in about 13% at 12 years (~1%/year), contributing substantially to the IDFS rates. For women Conclusions: The current updated analysis confirms findings from the original primary analysis that ET is non-inferior to CET in HR-positive, HER2-negative, node-negative EBC and a RS 11-25. As in the original primary analysis, the subgroup of women Citation Format: Joseph Sparano, Robert J. Gray, Della Makower, Kathy S. Albain, Daniel F. Hayes, Charles Geyer, Elizabeth Dees, Matthew P. Goetz, John A. Olson Jr, Tracy G. Lively, Sunil Badve, Thomas Saphner, Lynne I. Wagner, Timothy Whelan, Virginia Kaklamani, George Sledge. Trial Assigning Individualized Options for Treatment (TAILORx): An Update Including 12-Year Event Rates [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS1-05.

Introduction: Breast cancer treatment is associated with cancer-related cognitive impairment (CRCI). However, the differential effect of endocrine therapy (ET) vs chemotherapy followed by endocrine therapy (CET), including the impact of menopausal status, on CRCI is not well understood. Methods: Participants (pts) with hormone receptor positive, HER2 negative breast cancer with 1-3 positive lymph nodes and an Oncotype DX recurrence score of 0-25 enrolled in the RxPONDER trial were randomly assigned to ET alone versus CET. Until the health-related quality of life (HRQoL) accrual goal was reached, English speaking pts in the US were invited to complete HRQoL questionnaires including the 8-item PROMIS Perceived Cognitive Function Concerns (PCF) Short Form questionnaire shortly after randomization (baseline), as well as 6, 12, and 36 months after baseline. Analysis of measures of anxiety and fatigue is presented separately. Standardized T scores (mean 50; SD 10) for PCF were computed with higher scores indicating less cognitive impairment. The primary endpoint of this exploratory analysis was to compare mean PCF T scores by treatment arm and menopausal status. Separately by menopausal status, a generalized estimating equations (GEE) model was fit to the three timepoints adjusting for baseline to estimate the difference between treatment arms and whether there was a time trend over the three follow-up measures. Results: The HRQoL accrual exceeded the goal of 500 patients, with 74% of pts participating voluntarily until the QOL invitation was removed from the protocol (Dec 1, 2012). A total of 139 pre and 429 postmenopausal pts completed the questionnaires at baseline. T scores were similar between ET and CET arms at baseline [Table 1]. In the ET arm, T scores decreased from baseline to 6 and 12 months but recovered to baseline at 36 months. In the CET arm, T scores decreased from baseline to 6 months and 12 months but did not return to baseline at 36 months. The mean score difference between CET and ET over time was -3.02 (p=0.01) and -2.36 (p=0.003) for pre and postmenopausal pts, respectively. Adjusting for baseline, there was no significant time trend over the three follow-up periods for either premenopausal (p=0.12) or postmenopausal (p=0.49) pts. Dropoff occurred over time with 79%, 76%, 60% of pts at baseline participating at 6, 12, and 36 months. Complete endocrine treatment adherence data are not yet available at each timepoint. Conclusion: Chemoendocrine therapy has a greater negative effect on patient-reported CRCI compared to ET alone in both pre- and post-menopausal pts and it is sustained over 36 months. Interventions to prevent or treat CRCI are needed to improve the long-term quality of life of patients treated with CET. Table 1. Comparisons of mean Cognitive Function score by treatment arm and menopausal status. Citation Format: Irene Kang, Jamie K. Forschmiedt, Michelle M. Loch, William E. Barlow, Danika L. Lew, Julie R. Gralow, Funda Meric-Bernstam, Kathy S. Albain, Daniel F. Hayes, Nancy U. Lin, Edith A. Perez, Lori J. Goldstein, Priya Rastogi, Anne F. Schott, Steven Shak, Priyanka Sharma, Jieling Miao, Debu Tripathy, Lajos Pusztai, Gabriel N. Hortobagyi, Kevin Kalinsky, N. Lynn Henry. Patient-reported cognitive impairment in women participating in the RxPONDER trial (SWOG S1007) by menopausal status [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS1-04.

Background: Adjuvant chemotherapy is a common treatment for breast cancer patients with aggressive tumors, but imposes severe side effects. Although lymph node involvement is associated with higher likelihood of mortality, a subset of LN+ patients can survive without relapse, even without adjuvant chemotherapy. SWOG-8814 (S8814) was a randomized clinical trial of hormone receptor positive, LN+ post-menopausal women (pathologic stage T1-3N1-2) to compare patient outcome between endocrine therapy alone (tamoxifen), chemotherapy (cyclophosphamide, doxorubicin, and 5-fluorouracil) followed by 5 years of tamoxifen, and chemotherapy with concurrent tamoxifen. In this work, we evaluated the ability of a machine learning approach called multiple instance learning (MIL) to predict long-term overall (OS) and disease-free survival (DFS) in patients from the S8814 clinical trial via analysis of digitized H&E tissue slides. Methods: The training set (St, n=121) consisted of digitized H&E Whole Slide Images (WSIs) of ER+ LN+ patients from ECOG-2197 - a clinical trial to compare patient outcome under chemotherapy with doxorubicin/docetaxel vs. doxorubicin/cyclophosphamide. For each WSI, the nuclei of ten tumor patches (2000 × 2000 pixels) were automatically segmented by a pretrained machine learning algorithm called Convolutional Neural Networks (CNN). The results of the CNN’s segmentations were used to obtain ten corresponding feature vectors of 3963 features relating to nuclear morphology and spatial arrangement. Five top discriminative features (Haralick texture, Delaunay triangulation, orientation entropy, perimeter kurtosis, architecture) were identified by reliefF-MI feature filtering and forward selection on St. A Normalized Set Kernel Support Vector Machine classifier was trained on St to construct a MIL binary classifier (M+), which generates a continuous survival score to predict which patients would have >10-years of DFS and OS. The score cutoff thatmaximized F1 score on St was used to generate binary predictions of patient outcome. Blinded validation of the risk scores on S8814 was performed by SWOG. Results: M+ was significantly prognostic of OS in univariate analysis (HR=0.72, p=0.020, 95% CI= 0.55 – 0.95) on S8814. M+ stratifications remained statistically significant after multivariable analysis controlling for treatment, number of positive lymph nodes, and tumor size (HR = 0.71, p=0.017, 95% CI=0.54-0.94). M+ predictions did not achieve statistical significance for DFS in univariate analysis (HR=0.81, p=0.081, 95% CI 0.64-1.03) nor multivariable analysis (HR=0.81, p=0.080, 95% CI 0.63-1.03). M+ was not predictive of chemotherapy benefit for OS (p=0.88) and DFS (p=0.65). There was no correlation of the generated continuous score with Recurrence Score (r=-0.05), previously shown to be a predictive factor for chemotherapy benefit. Conclusion: Our findings demonstrate that nuclear morphology provides insight into overall survival in ER+ LN+ breast cancer patients. Future work will involve combining the MIL based morphologic predictor with other clinicopathologic factors to improve DFS risk stratification. Funding: Funding NIH/NCI grants U10CA180888, U10CA180819, U24CA196175; and in part by Genomic Health, INC. (now Exact Sciences Corp.) Citation Format: Daniel Shao, William E. Barlow, Haojia Li, Cheng Lu, Kathy S. Albain, James Rae, Daniel F. Hayes, Andrew K. Godwin, Alastair M. Thompson, Anant Madabhushi, Lajos Pusztai. Computer analysis of nuclear morphology with Multiple Instance Learning Predicts Overall Survival for Node Positive Breast Cancer Patients from SWOG S8814: A Blinded Validation Study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-11.

Objective Aromatase inhibitors (AI) are commonly used to treat hormone receptor positive (HR+) breast cancer. AI-induced musculoskeletal syndrome (AIMSS) is a common toxicity that causes AI treatment discontinuation. The objective of this genome-wide association study (GWAS) was to identify genetic variants associated with discontinuation of AI therapy due to AIMSS and attempt to replicate previously reported associations. Methods In the Exemestane and Letrozole Pharmacogenetics (ELPh) study, postmenopausal patients with HR+ non-metastatic breast cancer were randomized to letrozole or exemestane. Genome-wide genotyping of germline DNA was conducted followed by imputation. Each imputed variant was tested for association with time-to-treatment discontinuation due to AIMSS using a Cox proportional hazards model assuming additive genetic effects and adjusting for age, baseline pain score, prior taxane treatment, and AI arm. Secondary analyses were conducted within each AI arm and analyses of candidate variants previously reported to be associated with AIMSS risk. Results Four hundred ELPh participants were included in the combined analysis. Two variants surpassed the genome-wide significance level in the primary analysis (p-value−8), an intronic variant (rs79048288) within CCDC148 (HR=4.42, 95% CI: 2.67-7.33) and an intergenic variant (rs912571) upstream of PPP1R14C (HR=0.30, 95% CI: 0.20-0.47). In the secondary analysis, rs74418677, which is known to be associated with expression of SUPT20H, was significantly associated with discontinuation of letrozole therapy due to AIMSS (HR=5.91, 95% CI: 3.16-11.06). We were able to replicate associations for candidate variants previously reported to be associated with AIMSS in this cohort, but were not able to replicate associations for any other variants previously reported in other patient cohorts. Conclusions Our GWAS findings identify several candidate variants that may be associated with AIMSS risk from AI generally or letrozole specifically. Validation of these associations in independent cohorts is needed before translating these findings into clinical practice to improve treatment outcomes in patients with HR+ breast cancer.

Supplementary Table 1. Patients with high sTK1 and relative median sTK1 at different time points.

Supplementary Figure 2: Paclitaxel Exposure in Patients Who Did or Did Not Experience Peripheral Neuropathy-Induced Treatment Disruption. Patients who experienced PN-induced treatment disruption had greater median paclitaxel Tc>0.05 than patients who did not (11 hours (IQR 10-15) vs. 9.5 hours (IQR= 9-11), p=0.001) (left panel). The mean Cmax for patients who experienced treatment disruption was not statistically different from those who did not (2591.8 ng/mL (SD 897.9) vs. 2267.4 ng/mL (SD 521.2), p=0.18). The line in the box is the median. The lower and upper hinges correspond to the first and third quartiles and the lines extend 1.5 x IQR, representing an approximate 95% confidence interval.

Purpose:Metastasis requires malignant cell circulation from the primary to a distant tissue. Elevated levels of circulating tumor cells (CTC) portend a poor prognosis in breast and other cancers. Recent studies have suggested that CTC clusters may be a factor in the metastatic process. We conducted a prospective retrospective study of the SWOG0500 clinical trial to test whether CTC clusters are associated with poorer prognosis.Experimental Design:CTC CellSearch galleries from SWOG0500 trial were reread using prespecified criteria for CTC clusters, doublets, and enumeration. Survival analysis methods include Kaplan–Meier plots and log-rank tests.Results:Patients were classified into three prognostic subgroups based on baseline CTC/7.5 mL whole blood (WB): Arm A: P < 0.0001). Furthermore, doublets or clusters were significantly more common in patients who were ultimately assigned to Arm C versus B (54% vs. 25%; P < 0.0001). In Arm C, doublets and clusters were associated with worse overall survival than only doublets, clusters, or no doublets nor clusters at baseline (P = 0.008) and first follow-up (P = 0.010). When compared with enumeration alone, doublets, clusters, or both were not prognostic in patients who had 5–19 or ≥20 CTC/7.5 mL WB.Conclusions:In patients with metastatic breast cancer starting first-line chemotherapy, mortality is independent of the presence of CTC clusters, but rather depends on the number of CTC/7.5 mL WB.

PDF file - 46K, Patient Level Data. This table tabulates chemotherapy cycles received, response and DLT per patient.

Background: Endocrine therapy (ET) fails to induce a response in one half of patients with hormone receptor (HR)–positive metastatic breast cancer (MBC), and almost all will eventually become refractory to ET. Circulating tumor cells (CTC) are associated with worse prognosis in patients with MBC, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR-positive MBC.Methods: The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC.Results: The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in patients with MBC. CTC-ETI was successfully determined in 44 of 50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Interobserver concordance of CTC-ETI determination was from 94% to 95% (Kappa statistic, 0.90–0.91). Inter- and cell-to-cell intrapatient heterogeneity of expression of each of the CTC markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissues.Conclusions: CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC biomarker expression, are being evaluated in an ongoing prospective trial. Clin Cancer Res; 21(11); 2487–98. ©2014 AACR.See related commentary by Mathew et al., p. 2421

Describes the supplementary figures 1-10

This file contains supporting information of methods including compound syntheses, molecular modeling, reagents, proteomic & transcriptome profiling and QPCR primers.

Supplementary Figures S1, S2, S3 and Tables S1, S2

Sup. Table 1 depicts the synergism between MELK KD and RT in animal models, Sup. Table 2 shows the patient characteristics from the Wang dataset, Sup. Table 3 shows the patient characteristics from the Servant dataset

Purpose: Accumulating evidence supports the existence of breast cancer stem cells (BCSC), which are characterized by their capacity to self-renew and divide indefinitely and resistance to conventional therapies. The Notch pathway is important for stem cell renewal and is a potential target for BCSC-directed therapy.Experimental Design: Using human breast tumorgraft studies, we evaluated the impact of gamma secretase inhibitors (GSI) on the BCSC population and the efficacy of combining GSI with docetaxel treatment. The mouse experimental therapy paralleled a concurrent clinical trial in patients with advanced breast cancer, designed to determine the maximum-tolerated dose of the GSI, MK-0752, administered sequentially with docetaxel, and to evaluate BCSC markers in serial tumor biopsies.Results: Treatment with GSI reduced BCSCs in MC1 and BCM-2147 tumorgrafts by inhibition of the Notch pathway. GSI enhanced the efficacy of docetaxel in preclinical studies. In the clinical trial, 30 patients with advanced breast cancer were treated with escalating doses of MK-0752 plus docetaxel. Clinically, meaningful doses of both drugs were possible with manageable toxicity and preliminary evidence of efficacy. A decrease in CD44+/CD24−, ALDH+, and mammosphere-forming efficiency were observed in tumors of patients undergoing serial biopsies.Conclusions: These preclinical data show that pharmacologic inhibition of the Notch pathway can reduce BCSCs in breast tumorgraft models. The clinical trial shows feasibility of combination GSI and chemotherapy, and together these results encourage further study of Notch pathway inhibitors in combination with chemotherapy in breast cancer. Clin Cancer Res; 19(6); 1512–24. ©2012 AACR.

Supplementary Figure from A Randomized Trial of Fulvestrant, Everolimus, and Anastrozole for the Front-line Treatment of Patients with Advanced Hormone Receptor–positive Breast Cancer, SWOG S1222

MELK expression (by SAGE) in all normal and cancerous human tissues

Supplementary Figures: Supplementary Figure 1. Summary of duration on AZD9496 therapy, previous therapies, and circulating biomarkers status at baseline for each patient ranked by ascending dose; Supplementary Figure 2. Tumor pharmacodynamics. Supplementary Tables: Supplementary Table 1. Study Plan showing time-points of collection of biosamples for exploratory research; Supplementary Table 2. ddPCR primers and probes;Supplementary Table 3. Antibodies and conditions for IHC analysis Antibodies for IHC analysis; Supplementary Table 4. Patients with {greater than or equal to}1 CTC/7.5 mL WB within 120 days of fulvestrant wash-out period prior C1D1.

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients.Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110–22. ©2017 AACR.

Sup. Fig. 6 depicts proliferation with MELK pertubation, Sup. Fig. 7 depicts apoptosis by cleaved PARP, Sup. Fig. 8 depicts apoptosis by flow cytometry

Sup. Fig. 9 depicts GSEA analysis nominating DNA repair as a mechanism, Sup. Fig. 10 depicts molecular concept mapping (MCM) of MELK regulated genes

Purpose: While effective targeted therapies exist for estrogen receptor–positive and HER2-positive breast cancer, no such effective therapies exist for triple-negative breast cancer (TNBC); thus, it is clear that additional targets for radiosensitization and treatment are critically needed.Experimental Design: Expression microarrays, qRT-PCR, and Western blotting were used to assess MELK RNA and protein expression levels. Clonogenic survival assays were used to quantitate the radiosensitivity of cell lines at baseline and after MELK inhibition. The effect of MELK knockdown on DNA damage repair kinetics was determined using γH2AX staining. The in vivo effect of MELK knockdown on radiosensitivity was performed using mouse xenograft models. Kaplan–Meier analysis was used to estimate local control and survival information, and a Cox proportional hazards model was constructed to identify potential factors impacting local recurrence-free survival.Results: MELK expression is significantly elevated in breast cancer tissues compared with normal tissue as well as in TNBC compared with non-TNBC. MELK RNA and protein expression is significantly correlated with radioresistance in breast cancer cell lines. Inhibition of MELK (genetically and pharmacologically) induces radiation sensitivity in vitro and significantly delayed tumor growth in vivo in multiple models. Kaplan–Meier survival and multivariable analyses identify increasing MELK expression as being the strongest predictor of radioresistance and increased local recurrence in multiple independent datasets.Conclusions: Here, we identify MELK as a potential biomarker of radioresistance and target for radiosensitization in TNBC. Our results support the rationale for developing clinical strategies to inhibit MELK as a novel target in TNBC. Clin Cancer Res; 22(23); 5864–75. ©2016 AACR.

Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection.Experimental Design: Tumor tissue positive for HER2mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2mut).Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31–74) years and three (2–10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%–55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8–31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%–94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%–100%). In addition, ctDNA HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression.Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687–95. ©2017 AACR.

Purpose:Common resistance mechanisms to endocrine therapy (ET) in estrogen receptor (ER)–positive metastatic breast cancers include, among others, ER loss and acquired activating mutations in the ligand-binding domain of the ER gene (ESR1LBDm). ESR1 mutational mediated resistance may be overcome by selective ER degraders (SERD). During the first-in-human study of oral SERD AZD9496, early changes in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) were explored as potential noninvasive tools, alongside paired tumor biopsies, to assess pharmacodynamics and early efficacy.Experimental Design:CTC were enumerated/phenotyped for ER and Ki67 using CellSearch in serial blood draws. ctDNA was assessed for the most common ESR1LBDm by droplet digital PCR (BioRad).Results:Before starting AZD9496, 11 of 43 (25%) patients had ≥5 CTC/7.5 mL whole blood (WB), none of whom underwent reduction to +, two of whom had CTC-ER+ reduction. CTC-Ki67 status did not change appreciably. Patients with ≥5 CTC/7.5 mL WB before treatment had worse progression-free survival (PFS) than patients with P = 0.0003). Fourteen of 45 (31%) patients had ESR1LBDm+ ctDNA at baseline, five of whom had ≥2 unique mutations. Baseline ESR1LBDm status was not prognostic. Patients with persistently elevated CTC and/or ESR1LBDm+ ctDNA at C1D15 had worse PFS than patients who did not (P = 0.0007).Conclusions:Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER+ and ESR1LBDm+ ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker.

Table S1. Antibodies used for CTC and tissue studies. Table S2. Stage 2 stopping rules for the cumulative number of successfully assayed patients. Table S3. A. CTC-Enumeration Points based on CTC Enumeration B. CTC-Bio-Pointsa Footnotes:aOnly aliquots with CTC {greater than or equal to}5/7.5 ml WB evaluated bLow or negative ER expression is weighed more than BCL-2, HER2, and Ki67 due to its fundamental role in endocrine responsiveness. C. Calculation of CTC-ETI Scores. D. CTC-ETI Categories. Footnote: Abbreviations: CTC= circulating tumor cells; ER = estrogen receptor; ET= Endocrine Therapy; MBC = metastatic breast cancer; ET= endocrine therapy. Table S4. CTC-ETI determination in cultured human breast cancer cell lines spiked into normal human blood and evaluated by the CellSearch® system. Table S5. Examples of low, intermediate, and high CTC-ETI from patient samples. A. LOW CTC-ETI Patient # 5 (Primary Tissue: ER 90% positive, HER2 negative by FISH; Metastatic tissue: ER positive, HER2 negative by FISH)* B. INTERMEDIATE CTC-ETI Patient #4 (Primary Tissue: ER {greater than or equal to}95% positive, HER2 0 by IHC; Metastatic tissue: insufficient tissue to evaluate ER and HER2)* C. HIGH CTC-ETI Patient # 3 (Primary Tissue: ER 60% positive, HER2 1+/2+ by IHC; Metastatic tissue: ER 75% positive; HER2 1+ by IHC)* Footnote: *ER and HER2 as per initial clinical laboratory using the laboratory's criteria for positivity or negativity. Table S6.< CTC-ETI for each patient. Footnote: aAF =Analytical Failure (CTCbTF=Technical Failure (Machine failure in at least one of 4 aliquots). Table S7. Concordance between biomarkers in primary and metastatic tissues and CTC. aKappa score for concordance between CTC and tissue biomarker for respective biomarker.

Purpose:Serum thymidine kinase 1 (sTK1) activity is associated with poor prognosis in metastatic breast cancer (MBC). We assessed the prognostic effect of sTK1 in patients with hormone receptor–positive MBC treated on a prospective randomized trial of anastrozole (A) versus A plus fulvestrant (A + F).Patients and Methods:sTK1 was assessed in 1,726 serums [baseline (BL), cycles 2, 3, 4, and 7] using the DiviTum assay. A prespecified cutoff of ≥200 Du/L was considered high. Progression-free survival (PFS) and overall survival (OS) were analyzed by Kaplan–Meier, log-rank tests, and Cox regression.Results:BL sTK1 was elevated in 171 (40%) of 432 patients. Patients with high versus low BL sTK1 had significantly worse PFS [median 11.2 vs. 17.3 months, HR = 1.76; 95% confidence interval (CI; 1.43–2.16); P < 0.0001] and OS [median 30 vs. 58 months, HR = 2.38; 95% CI (1.91–2.98); P < 0.0001]. OS was significantly better for patients with high sTK1 who did not have prior adjuvant tamoxifen and who received A + F versus A alone [median 46 vs. 21 months, HR = 0.58; 95% CI (0.38–0.87); P = 0.0087]. Patients with low sTK1 had no difference in outcomes by therapy (P = 0.44). At serial timepoints, high versus low sTK1 had significantly worse subsequent PFS and OS [at cycle 2: PFS HR = 1.70, 95% CI (1.34–2.17); P < 0.0001, OS HR = 2.51, 95% CI (1.93–3.26); P < 0.0001].Conclusions:High sTK1 at BL and subsequent timepoints is associated with worse prognosis in patients with MBC starting first-line endocrine therapy (ET). Patients with low sTK1 at BL have comparable outcomes on single-agent or combination ET.

Purpose: Paclitaxel exposure, specifically the maximum concentration (Cmax) and amount of time the concentration remains above 0.05 μmol/L (Tc>0.05), has been associated with the occurrence of paclitaxel-induced peripheral neuropathy. The objective of this study was to validate the relationship between paclitaxel exposure and peripheral neuropathy.Experimental Design: Patients with breast cancer receiving paclitaxel 80 mg/m2 × 12 weekly doses were enrolled in an observational clinical study (NCT02338115). Paclitaxel plasma concentration was measured at the end of and 16–26 hours after the first infusion to estimate Cmax and Tc>0.05. Patient-reported peripheral neuropathy was collected via CIPN20 at each dose, and an 8-item sensory subscale (CIPN8) was used in the primary analysis to test for an association with Tc>0.05. Secondary analyses were conducted using Cmax as an alternative exposure parameter and testing each parameter with a secondary endpoint of the occurrence of peripheral neuropathy–induced treatment disruption.Results: In 60 subjects included in the analysis, the increase in CIPN8 during treatment was associated with baseline CIPN8, cumulative dose, and relative dose intensity (P < 0.05), but neither Tc>0.05 (P = 0.27) nor Cmax (P = 0.99). In analyses of the secondary endpoint, cumulative dose (OR = 1.46; 95% confidence interval (CI), 1.18–1.80; P = 0.0008) and Tc>0.05 (OR = 1.79; 95% CI, 1.06–3.01; P = 0.029) or Cmax (OR = 2.74; 95% CI, 1.45–5.20; P = 0.002) were associated with peripheral neuropathy–induced treatment disruption.Conclusions: Paclitaxel exposure is predictive of the occurrence of treatment-limiting peripheral neuropathy in patients receiving weekly paclitaxel for breast cancer. Studies are warranted to determine whether exposure-guided dosing enhances treatment effectiveness and/or prevents peripheral neuropathy in these patients. Clin Cancer Res; 24(15); 3602–10. ©2018 AACR.

Supplemental Figure 1 (A) Progression-Free Survival in S0226 (B) Overall Survival in S0226

Supplementary table 1. Findings of multivariable linear regressions analyzing genetic associations between candidate gene SNPs and lipid profiles in exemestane-treated participants complementary to statistically significant findings in letrozole-treated participants. Supplementary table 2. Findings of multivariable linear regressions analyzing genetic associations between candidate gene SNPs and lipid profiles in only Caucasian women taking both letrozole and exemestane complementary to statistically significant findings in letrozole-treated participants. *new significant finding (in letrozole only), and corresponding data for exemestane (additive rs1062033 and dominant rs1062033 models were not included due to number of homozygotes

Purpose: Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). We tested whether EpCAM-based capture system (CellSearch) is effective in patients with triple-negative (TN) MBC, and whether CTC apoptosis and clustering enhances the prognostic role of CTC.Experimental Design: CTC enumeration and apoptosis were determined using the CXC CellSearch kit at baseline and days 15 and 29 in blood drawn from TN MBC patients who participated in a prospective randomized phase II trial of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) with or without tigatuzumab. Association between levels of CTC and patient outcomes was assessed using logistic regression, Kaplan–Meier curves, and Cox proportional hazards modeling.Results: Nineteen of 52 (36.5%), 14 of 52 (26.9%), and 13 of 49 (26.5%) patients who were evaluable had elevated CTC (≥5 CTC/7.5 mL whole blood) at baseline and at days 15 and 29, respectively. Patients with elevated versus not elevated CTC at each time point had worse progression-free survival (PFS; P = 0.005, 0.0003, 0.0002, respectively). The odds of clinical benefit response for those who had elevated versus low CTC at baseline and days 15 and 29 were 0.25 (95% CI: 0.08–0.84; P = 0.024), 0.19 (95% CI: 0.05–0.17; P = 0.014), and 0.06 (95% CI: 0.01–0.33; P = 0.001), respectively. There was no apparent prognostic effect comparing CTC apoptosis versus non-apoptosis. Presence of CTC cluster at day 15 and day 29 was associated with shorter PFS.Conclusions: CTC were detected using CellSearch assay in approximately one-third of TN MBC patients. Elevated CTC at baseline and days 15 and 29 were prognostic, and reductions in CTC levels reflected response. Clin Cancer Res; 21(12); 2771–9. ©2015 AACR.See related article by Forero-Torres et al., p. 2722

Purpose:Metastatic hormone receptor (HR)-positive, HER2-negative breast cancer is an important cause of cancer mortality. Endocrine treatment with or without additional targeted therapies has been the mainstay of treatment. This trial was designed to evaluate the combination of fulvestrant plus everolimus versus fulvestrant, everolimus, and anastrozole compared with fulvestrant alone in the first-line treatment of advanced HR-positive, HER2-negative breast cancer.Patients and Methods:This randomized placebo-controlled trial included postmenopausal women with HR-positive, HER2-negative advanced breast cancer who had received no prior systemic therapy for metastatic disease. Participants were randomized to one of three treatment arms and the primary outcome was progression-free survival (PFS), comparing combinations of fulvestrant and everolimus with or without anastrozole with fulvestrant alone. Circulating tumor cells (CTC), as measured with two different methods, and circulating tumor DNA (ctDNA) were evaluated serially prior to treatment and the beginning of the second cycle of therapy.Results:Due in part to changes in clinical practice, the study was closed after accruing only 37 participants. There was no evidence that everolimus-containing combination treatment improved PFS or overall survival relative to fulvestrant alone. When modeled continuously, an association was observed of baseline CTC and ctDNA with poorer survival.Conclusions:Although power of the study was limited, the findings were unable to support the routine use of everolimus combination endocrine therapy in the first-line treatment of advanced hormone-sensitive breast cancer. Prognostic impact of baseline ctDNA and copy-number variations in CTC was demonstrated.

Supplementary Figure 1-5. Supplementary Figure 1. Example of cluster image using the CellSearch® system. Supplementary Figure 2. REMARK diagram of the 19 patients who had elevated CTC (Group B/C) at baseline that were used to study "CTC-response". Supplementary Figure 3. PFS for TIG-treated patients versus the control group according to baseline CTC levels: CTC