Background: New strategies to enhance endocrine therapy (ET) efficacy and/or overcome resistance by targeting key survival pathways are needed. Preclinical data indicate that unwanted effects of ET include reactivation of the Notch pathway, critical for breast tumor initiating (stem) cells. Notch inhibition with gamma secretase inhibitors (GSI) enhances tamoxifen (tam) efficacy in xenografts, but impact of GSI+ET in human breast cancer (BC) is unknown. Our objective was to add short exposure of the GSI MK-0752 to ongoing tam or letrozole (let) in the presurgical window to assess feasibility, safety and biomarker/pathway impact in a 20-patient (pt) pilot study (ClinTrials.gov NCT00756717). We previously evaluated several biomarkers in the first cohort, which showed promise with Notch and proliferation inhibition. We present new results adding the final cohort, plus additional biomarkers and microarray analyses. Methods: Pts with early stage ER+ BC received 25 days (d) of ET. MK-0752 was added d15 (350 mg PO 3d on, 4d off, 3d on) with definitive surgery d25. Core biopsies were done at baseline, d14 and d25, with qRT-PCR for Notch-related and other genes critical to stem cell renewal/proliferation. Gene expression levels after GSI (d25) vs ET alone (d14) were analyzed and d25 changes in all pts combined for each gene were compared. Microarray expression estimates and modeling were performed using dCHip and Red-R, implementing gene-wise comparisons using Limma. Probes were defined as significantly regulated by paired t tests if p ≤ 0.001 for the comparisons of baseline to tam/let and tam/let to tam/let+GSI. Data were exploratory so all probe data were included in the modeling, and no corrections for multiple comparisons were used. Differentially expressed genes were submitted to DAVID for pathway analysis. Results: Of 22 pts accrued, 20 (11 tam, 9 let) were evaluable, meeting accrual goals (2 withdrew before MK-0752); 19 completed therapy to date. Toxicity was minimal. Significant (p<.05) changes in mRNA levels after GSI+ET vs end of ET in 17 pts (3 in progress) were down-regulation of Notch4 in 13; Ki67, 13; Notch1, 12; RUNX1 (stem cell transcription factor), 13; ADAM19 (disintegrin/metalloproteinase), 12; MMP7 (Wnt target), 11; CCND1 (cyclin D1), 10; and up-regulation of NOXA (pro-apoptotic BH3-only gene), 13. Microarray analyses (10 completed, remainder underway) found significant numbers of GSI-regulated genes that were independent of tam/let. Of 4036 genes increased by GSI, 2777 were unchanged by tam/let; of 3978 genes decreased by GSI, 1017 were not impacted by tam/let. For example, of genes regulated by GSI alone, there was modulation of important cancer pathways: Wnt5a, FGFs, FGFR, IGF-1R were decreased; Fas and caspases were increased. These changes in gene expression are being compared with ET resistance profiles. Conclusions: Short exposure of MK-0752 added to ET was feasible, well tolerated, and resulted in significant biomarker response in all tumors. MK-0752 favorably modulated proliferation, apoptosis, stem cell and metastasis-related targets, and impacted critical cancer pathways. This suggests potential roles for MK-0752 in optimizing endocrine therapy and overcoming endocrine resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-5.
Background: Resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) for ER+ breast cancer is a major cause of mortality and new treatment paradigms are needed. Cancer stem cells drive breast cancer growth and are resistant to standard therapy. Notch signaling aids survival of these resistant stem cells and is inhibited by gamma-secretase inhibitors (GSI). We showed combining GSI with ET in mice caused shrinkage of breast cancer tumors. A presurgical window biomarker modulation model was used to confirm this discovery in humans. Methods: The GSI MK-0752 was added to ET in patients before definitive surgery (ClinTrials.gov NCT00756717). There were 3 biopsies: day 0 (prestudy), day 14 (after ET alone), and day 25 at definitive surgery (after continued ET plus MK-0752, 350 mg orally 3d on, 4d off, 3d on). Biopsies were analyzed for genes increased or decreased by GSI, to confirm that Notch and cancer stem cell pathways were inhibited. Real-time PCR was used to validate expression of genes identified in pathway analyses of microarray datasets generated from the biopsies. Mammosphere-forming assays were performed to confirm that ET+GSI impacts breast cancer stem cells. The qRT-PCR data were evaluated using ANOVA with repeated measures and ANOVA was performed on mammosphere results. Results: The accrual goal was met and therapy well-tolerated in 20 evaluable women (PSABCS 2011, abs# S1-5). Of 33 genes identified by analysis of expression microarrays, 19 genes (FDR<8%) were impacted significantly by GSI+ ET (3 increased, 16 decreased) compared to initial biopsy and/or ET alone. Genes with increased expression were DAXX, NOXA (both pro-apoptotic) and LNFG (tumor suppressor). Six of 16 genes that decreased (NOTCH1, NOTCH4, HEYL, HES1, HES5, and HEY2) are Notch pathway-associated genes. The GSI decreased expression of 3 genes from cell cycle and proliferative pathways (Ki67, CCND1, CCNA2) and inhibited 2 genes expressed in cancer stem cells (RUNX1 and ALDH1A1). Five genes directly/indirectly regulated by Notch were decreased by GSI (RICTOR, RPTOR, MMP7, ADAM19, and PRH). Estrogen deprivation for 3 days, mimicking short exposure to an AI, increased mammosphere-forming ability of ER+ breast cancer cells more than 2 fold. The GSI MRK-003 blocked this mammosphere formation by 95%-98%. Conclusions: A 7-day course of the GSI MK-0752 added to ET in the presurgical window had significant biomarker responses: decrease in Notch signaling, cancer stem cell genes, proliferation-associated genes, the mTORC1 and 2 complex genes RICTOR and RPTOR, metalloproteinases that promote metastasis, and PRH; as well as increase in 3 key genes that promote apoptosis and tumor suppression. These results suggest that 1) GSI inhibited the intended Notch pathway, 2) putative breast cancer stems cells can be targeted by this strategy, and 3) the biomarkers identified create a gene signature for anti-Notch therapy in ER+ breast cancer. Validation of efficacy of the GSI+ET therapy combination and this gene signature in a clinical trial is planned. Support: Breast Cancer Research Foundation (research grant), Merck Oncology (drug/arrays), Swim Across America (clinical trial costs), and DOD BC073237 (KRC). Citation Format: Kathy S Albain, Andrei Y Zlobin, Kyle R Covington, Brian T Gallahger, Susan G Hilsenbeck, Cheryl M Czerlanis, Shelly Lo, Patricia A Robinson, Ellen R Gaynor, Constantine Godellas, Davide Bova, Kathy Czaplicki, Barbara Busby, Patrick J Stiff, Suzanne AW Fuqua, Lucio Miele, Clodia Osipo. Identification of a notch-driven breast cancer stem cell gene signature for anti-notch therapy in an ER+ presurgical window model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S4-03.
Background: Breast tumor initiating cells (TIC) use Notch receptors/ligands with other pathways for self renewal, resulting in tumor proliferation and progression. We showed that Notch inhibition with gamma secretase inhibitors (GSI) potentiates the effects of tamoxifen (tam) in xenografts (Rizzo et al. Cancer Res 2008). It is unknown whether GSIs plus endocrine therapy result in modulation of Notch and other proliferation markers in human breast cancer. Our objective was to add short exposure of the GSI MK-0752 to ongoing tam or letrozole (letr) during the presurgical window to determine 1) feasibility, 2) safety/tolerance, and 3) impact on biomarkers. We report the initial cohort of this pilot study (ClinTrials. gov NCT00756717). Methods: Patients (pts) with early stage ERα + breast cancer were treated with 25 days of tam or letr. On day 15 MK-0752 was added to endocrine therapy (350 mg orally 3 days on, 4 days off, 3 days on), with definitive surgery day 25. Formalin fixed, paraffin embedded biopsies were obtained at baseline, day 14 and final surgery, with histologic confirmation of tumor content >50% and RNA extraction by standard methods. Q-PCR was done for Notch1, Notch3, Notch4, Deltex, Jagged1, c-myc, HEY1, HEY2, HES1, PS2, C-Myc, Cyclin A2, NOXA (pro-apoptotic protein), Ki67, Dicer-1, RPL13 (internal control). Ct averages for 3 replicates were used and mRNA levels were calculated by the 2ΔΔCt method. Baseline gene expression levels were used as comparators for days 14 and 25 levels in each pt. The first cohort of 10 pts was analyzed to determine if enough signals were present to justify expanding the cohort at this dose to 20 pts and possibly test a second cohort on an alternate MK-0752 dose/schedule. Results: The initial cohort of 10 pts completed all therapy (4 tam, 6 letr), all biopsies and definitive surgery on schedule. One other pt withdrew prior to starting MK-0752 due to hypertension. Toxicity was minimal: grade 1 periorbital edema/cough, nausea, and axillary paresthesias in 1 pt each; grade 1 facial rash, 2 pts; and grade 2 fatigue, 1 pt. There was no diarrhea or surgical complications. Significant changes occurred in molecular marker levels after MK-0752 plus tam/letr (day 25) vs. end of tam/letr alone (day 14) as follows: Ki67 mRNA decreased in 9/10 pts; Notch4 decreased, 10/10; NOXA increased, 6/10; and Notch1 decreased, 6/10. Other markers showed inter-individual variations and will be presented, along with results of the global gene expression profiling (in progress). Conclusions: The addition of a short exposure of the GSI MK-0752 to ongoing endocrine therapy was feasible, safe, and well tolerated in pts with ERα + early breast cancer prior to definitive surgery. It results in anti-proliferative and pro-apoptotic effects at the molecular level. Notch4, which plays a key role in breast TIC, was the most consistent molecular marker of response in this setting. This suggests a potential anti-TIC effect of this combination and a role in overcoming endocrine resistance. Accrual to the expanded cohort is underway. If findings are confirmed, the second study with alternate MK-0752 dose/schedule may commence. Funding: Swim Across America, Inc. (clinical trial costs); Merck (drug supply, profiling) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-12.
We present a sensitivity analysis approach with multiple methods for analyzing treatment effects on patient QOL in the presence of substantial, non-ignorable missing data in an advanced stage disease clinical trial. We conclude that the two treatment arms did not differ statistically in their effects on patient QOL over a 25-week treatment period.
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