Haddad, Tufia C.

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor–positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Abstract Background: NOTCH oncogenic signaling induces epithelial-to-mesenchymal transition (EMT) and tumor stemness that play a critical role in driving tumor progression. Following interaction of NOTCH receptors (NOTCH 1, 2, 3, and 4) with their ligands (DELTA-LIKE 1, 3, 4 and JAGGED 1, 2), γ- secretase complex performs an intra-membrane cleavage releasing the NOTCH intracellular domain (NNICD). NNICD translocates to the nucleus and induces the expression of HEY1 and HES1 transcription factors that in turn orchestrate NOTCH-mediated EMT and stemness reprogramming. Materials and Methods: Parental MCF-7 and unique metastatic MCF-7RAF-1 xenografts with constitutively active MAPK signaling (D’Assoro et al., Oncogene 2014: 33:599-610) were used to define the extent to which increased expression of NOTCH signaling was linked to distant metastases. SKY analysis was performed to assess the grade of chromosomal instability (CIN) in tumor xenografts. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of CD24 luminal marker was characterized by FACS analysis and immunofluorescence. Global transcriptome analysis was performed using Affymetrix® microarray. Highly invasive MDA-MB 231 breast cancer cells were used to perform a tail vein cancer cell-seeding assay. The LY411575 γ-secretase inhibitor (Pan-NOTCH inhibitor) was used to block NOTCH signaling in MDA-MB 231 cells. Results: Metastasis Initiating Cells (MICs) isolated from the lungs of nude mice carrying MCF-7RAF-1 xenografts exhibited a higher degree of genomic stability than primary tumor xenografts that was characterized by the absence of centrosome amplification and non-clonal chromosomal abnormalities. MICs also showed higher mammosphere forming capacity that was linked to lower expression of luminal marker CD24. Significantly, global transcriptome analysis showed that expression of NOTCH3 and HES1 stemness genes was increased in MICs. To corroborate the role of NOTCH signaling in inducing metastatic seeding, MDA-MB 231 breast cancer cells were pre-treated with 1 µM of LY411575 After 72 hours viable cells were injected into the tail vein of immune-compromised mice to develop lung metastases. While animals injected with untreated MDA-MB 231 cells developed lung metastases, those injected with the LY411575-treated MDA-MB 231 cells failed to develop lung metastatic lesions. Conclusions: These preliminary findings demonstrate the role of NOTCH stemness signaling in promoting metastatic seeding of breast cancer cells. Since NOTCH signaling is a “druggable target”, these results also provide the rationale to develop novel NOTCH-targeted therapies to inhibit tumor stemness and early onset of distant metastases. Citation Format: Alexey Leontovich, Jeffrey Salisbury, Candace L. Haddox, Lisa Mills, Tufia Haddad, James McCubrey, Matthew Goetz, James Ingle, Evanthia Galanis, Antonino B. D'Assoro. NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2017-4851

2 Background: Women with atypical hyperplasia (AH) on breast biopsy have an aggregate increased risk of breast cancer (BC), but accurate personalized risk prediction is desirable to facilitate individual clinical management decisions. Currently used models provide poor BC risk prediction for women with AH. Our goal was to develop and validate an improved risk prediction model for women with AH. Methods: From a cohort of 13,538 women with benign breast disease from 1967-2001, pathology review confirmed 699 with AH. Clinical risk factors and histologic features of the tissue biopsy were recorded, and BC events were ascertained from study questionnaires, tumor registry, and review of medical records. Using a lasso approach, 23 variables were assessed for model inclusion. Lasso-identified features were then fit in a Cox regression model to estimate BC risk. Model discrimination was assessed with C-statistics in the model-building set and in a separate external validation set. Calibration was assessed by comparing observed to predicted breast cancer counts. Results: The model-building set comprised 699 women with 142 BC events (median follow-up 8.1 years), and the external validation set comprised 461 women with 114 BC events (median follow-up 11.4 years). The final model included three covariates: age at biopsy, age squared, and number of foci of AH. Model performance was good, with a C-statistic of 0.622 (SE = 0.027) in the model-building set and 0.594 (SE = 0.029) in the external validation set. The model is well-calibrated, with observed to expected numbers of BCs nearly equal across all post-biopsy follow-up years. Conclusions: We propose a new model for predicting BC risk in women with AH based on age at biopsy and number of foci of atypia. This model provides absolute risk estimates for women with AH, has good discriminatory ability, is well-calibrated, and validates in an external cohort.

Abstract Background: Intrinsic resistance has been linked to ER down-regulation and poor clinical outcome in breast tumors. Aurora-A kinase (AURKA) promotes distant metastases through activation of epithelial to mesenchymal transition (EMT) programming and expansion of breast tumor initiating cells (BTICs) harboring a CD44high/CD24low phenotype. Moreover, AURKA induces down-regulation of ERα expression and resistance to endocrine therapy that is mediated by SMAD5 transcription factor (D’Assoro et al., Oncogene 2014: 33:599-610). The aim of this study was to evaluate the role of AURKA in endocrine resistance induced by breast cancer stemness and ER down-regulation. Materials and Methods: We employed parental MCF-7 and variant MCF-7 cells ex-vivo (vMCF- 7/LCC9, vMCF-7/FR) that have high endogenous levels of AURKA, are ERlow, and exhibit in vivo resistance to fulvestrant. Real-Time Cell Proliferation and Apoptosis assays were performed to assess response to endocrine and/or AURKA-targeted therapy using the innovative IncuCyte ZOOM System. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of ER, CD44 and CD24 markers was characterized by FACS analysis and immunofluorescence. Results: Pharmacologic inhibition of AURKA activity with the small molecule, alisertib, significantly restored sensitivity to fulvestrant in vMCF-7/LCC9 and vMCF-7/FR cells. To establish whether fulvestrant resistance was linked to enrichment of BTICs, breast cancer cells were cultured under non-adherent conditions to generate mammospheres. vMCF-7/FR cells generated mammospheres at higher efficiency compared to parental cells suggesting that vMCF-7/FR cells exhibited high self-renewal activity. vMCF-7/FR-derived mammospheres displayed a stem cell-like CD44high/CD24low phenotype that was further characterized by low levels of ER expression and resistance to fulvestrant. When vMCF-7 FR-derived mammospheres were treated with alisertib alone or combined with fulvestrant, the combination was more effective in mediating restoration of endocrine sensitivity. The phenotype of the alisertib-treated mammospheres was functionally linked to loss of CD44 expression, restoration of CD24 and ER expression, and sensitivity to fulvestrant. Conclusions: These findings provide further evidence that AURKA activity plays a central role in breast cancer progression through the genesis and clonal expansion of BTICs that are characterized by ER down-regulation and resistance to endocrine therapy. These results are important for the treatment of endocrine resistant breast tumors because they provide the rationale for the development of an innovative therapeutic approach to combine endocrine therapy with a targeted inhibitor of AURKA activity. Citation Format: Antonino B. D’Assoro, Tufia Haddad, Matthew Goetz, Mary Kuffel, John Hawse, Vera Suman, Jeffrey Salisbury, James Ingle, Evanthia Galanis. Pharmacologic targeting of Aurora-A kinase restores ERα expression and endocrine sensitivity through impairment of breast cancer stemness. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3313.

BACKGROUNDWomen with benign breast disease (BBD) have an increased risk of developing breast cancer (BC). Nearly 30% of all BCs develop in women with prior BBD. Information regarding features of the expected number of BCs after BBD would enhance individualized surveillance and prevention strategies for these women. In the current study, the authors sought to characterize BCs developing in a large cohort of women with BBD.METHODSThe current study cohort included 13,485 women who underwent breast biopsy for mammographic or palpable concerns between 1967 and 2001. Biopsy slides were reviewed and classified as nonproliferative disease, proliferative disease without atypia, or atypical hyperplasia. BCs were identified by follow‐up questionnaires, medical records, and Tumor Registry data. BC tissues were obtained and reviewed.RESULTSWith median follow‐up of 15.8 years, 1273 women developed BC. The majority of BCs were invasive (81%), of which 61% were ductal, 13% were mixed ductal/lobular, and 14% were lobular. Approximately two‐thirds of the BC cases were intermediate or high grade, and 29% were lymph node positive. Cancer characteristics were similar across the 3 histologic categories of BBD, with a similar frequency of ductal carcinoma in situ, invasive disease, tumor size, time to invasive BC, histologic type of BC, lymph node positivity, and human epidermal growth factor receptor 2 positivity. Women with atypical hyperplasia were found to have a higher frequency of estrogen receptor‐positive BC (91%) compared with women with proliferative disease without atypia (80%) or nonproliferative disease (85%) (P = .02).CONCLUSIONSA substantial percentage of all BCs develop in women with prior BBD. The majority of BCs after BBD are invasive tumors of ductal type, with a substantial number demonstrating lymph node positivity. Of all the BCs in the current study, 84% were estrogen receptor positive. Prevention therapy should be strongly encouraged in higher‐risk women with BBD. Cancer 2016;122:378–385. © 2015 American Cancer Society.

Background Axillary web syndrome (AWS) is a condition that may develop following breast cancer surgery and that presents as a palpable axillary cord of tissue. Objective The purposes of this study were: (1) to determine the clinical characteristics of AWS related to movement, function, pain, and postoperative edema and (2) to define the incidence of and risk factors for AWS within the first 3 months following breast cancer surgery. Design This was a prospective cohort study with a repeated-measures design. Methods Women who underwent breast cancer surgery with sentinel node biopsy or axillary lymph node dissection (N=36) were assessed for AWS, shoulder range of motion, function, pain, and postoperative edema (using girth measurements, bioimpedance, and tissue dielectric constant) at 2, 4, and 12 weeks. Demographic characteristics were used for risk analysis. Results Seventeen women (47.2%) developed AWS, and AWS persisted in 10 participants (27.8%) at 12 weeks. Abduction range of motion was significantly lower in the AWS group compared with the non-AWS group at 2 and 4 weeks. There were no differences between groups in measurements of function, pain, or edema at any time point. Trunk edema measured by dielectric constant was present in both groups, with an incidence of 55%. Multivariate analysis determined lower body mass index as being significantly associated with AWS (odds ratio=0.86; 95% confidence interval=0.74, 1.00). Limitations Limitations included a short follow-up time and a small sample size. Conclusion Axillary web syndrome is prevalent following breast/axilla surgery for early-stage breast cancer and may persist beyond 12 weeks. The early consequences include movement restriction, but the long-term effects of persistent AWS cords are yet unknown. Low body mass index is considered a risk factor for AWS.

AbstractThe many improvements in breast cancer therapy in recent years have so lowered rates of recurrence that it is now difficult or impossible to conduct adequately powered adjuvant clinical trials. Given the many new drugs and potential synergistic combinations, the neoadjuvant approach has been used to test benefit of drug combinations in clinical trials of primary breast cancer. A recent FDA-led meta-analysis showed that pathologic complete response (pCR) predicts disease-free survival (DFS) within patients who have specific breast cancer subtypes. This meta-analysis motivated the FDA's draft guidance for using pCR as a surrogate endpoint in accelerated drug approval. Using pCR as a registration endpoint was challenged at ASCO 2014 Annual Meeting with the presentation of ALTTO, an adjuvant trial in HER2-positive breast cancer that showed a nonsignificant reduction in DFS hazard rate for adding lapatinib, a HER-family tyrosine kinase inhibitor, to trastuzumab and chemotherapy. This conclusion seemed to be inconsistent with the results of NeoALTTO, a neoadjuvant trial that found a statistical improvement in pCR rate for the identical lapatinib-containing regimen. We address differences in the two trials that may account for discordant conclusions. However, we use the FDA meta-analysis to show that there is no discordance at all between the observed pCR difference in NeoALTTO and the observed HR in ALTTO. This underscores the importance of appropriately modeling the two endpoints when designing clinical trials. The I-SPY 2/3 neoadjuvant trials exemplify this approach. Clin Cancer Res; 21(13); 2911–5. ©2015 AACR.