Summary of Study ST003315

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002062. The data can be accessed directly via it's Project DOI: 10.21228/M82Z4P This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003315
Study Title	Randomized phase II trial of pre-operative fulvestrant with or without enzalutamide for ER+/Her2- primary breast cancer: effects on tumor immune microenvironment and clinical outcomes.
Study Summary	This randomized phase II trial of 4 months of neoadjuvant fulvestrant (Fulv) alone or with enzalutamide (Combo) assessed whether the addition of AR blockade to Fulv would limit residual tumor at time of surgery, as measured by modified preoperative endocrine predictive index (PEPI) score. Eligible patients were women with ER+/HER2- primary BC cT2 or greater. Lithium-heparin (LiHep) plasma samples were obtained every 4 weeks until surgery and one month after surgery then analyzed by MS-based metabolomics.
Institute	University of Colorado Anschutz Medical Campus
Last Name	Haines
First Name	Julie
Address	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email	julie.haines@cuanschutz.edu
Phone	3037243339
Submit Date	2024-06-28
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-08-01
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002062
Project DOI:	doi: 10.21228/M82Z4P
Project Title:	Randomized phase II trial of pre-operative fulvestrant with or without enzalutamide for ER+/Her2- primary breast cancer: effects on tumor immune microenvironment and clinical outcomes.
Project Summary:	Most ER+ breast cancers (BC) express androgen receptors (AR). This randomized phase II trial of 4 months of neoadjuvant fulvestrant (Fulv) alone or with enzalutamide (Combo) assessed whether the addition of AR blockade to Fulv would limit residual tumor at time of surgery, as measured by modified preoperative endocrine predictive index (PEPI) score. Eligible patients were women with ER+/HER2- primary BC cT2 or greater. Stratification factors were clinical node and T-stage. Fresh tumor biopsies were required at study entry, after 4 weeks on therapy (W5), and at surgery (W17). Laboratory analyses on tumors included immunochemistry (IHC) for ER/PR/AR/GR and Ki67 protein, evaluation of gene expression, multiplex for myeloid lineage immune cells, reverse phase protein array, and plasma metabolomic analyses. 59 out of 69 consented patients were evaluable. Toxicity was as expected with endocrine therapy. Combo achieved PEPI=0 more frequently (24%: 8/33) than Fulv (8%: 2/26). Ki67 was <10% across arms by W5 or surgery in most patients (23/33 (70%) on Combo, 21/26 (81%) on Fulv). mTOR activation was elevated in tumors with poor Ki67 response. Tumors in both arms showed decreased estrogen-regulated and cell division gene sets, while Combo uniquely exhibited enrichment of immune activation genes sets, including interferon gamma, complement, inflammation, antigen processing, and B and T cell activation. Multiplex IHC showed significantly reduced tumor-associated macrophages and CD14+/HLADR-/CD68- MDSCs with Combo at W5. In summary, Combo achieved a higher PEPI=0 response, Ki67 response, and more activated tumor immune microenvironment than Fulv. The odds of response were 4.6-fold higher for patients with ILC versus IDC.
Institute:	University of Colorado Anschutz Medical Campus
Laboratory:	Core director Angelo D'Alessandro, study PI Jennifer Richer
Last Name:	Haines
First Name:	Julie
Address:	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email:	julie.haines@cuanschutz.edu
Phone:	3037243339

Subject:

Subject ID:	SU003436
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Histology	Lab Ki67 Group	PEPI Score	Visit	Arm
SA359418	AG-54	Plasma	Ductal	High BL, High W5	>0	Baseline	Combination
SA359419	AG-45	Plasma	Ductal	High BL, High W5	>0	Baseline	Combination
SA359420	AG-80	Plasma	Ductal	High BL, High W5	>0	Baseline	Combination
SA359421	AG-72	Plasma	Ductal	High BL, High W5	>0	Baseline	Fulvestrant
SA359422	AG-98	Plasma	Ductal	High BL, High W5	>0	Baseline	Fulvestrant
SA359423	AG-28	Plasma	Ductal	High BL, High W5	>0	Baseline	Fulvestrant
SA359424	AG-109	Plasma	Ductal	High BL, High W5	>0	Baseline	Fulvestrant
SA359425	AG-81	Plasma	Ductal	High BL, High W5	>0	C4D1	Combination
SA359426	AG-55	Plasma	Ductal	High BL, High W5	>0	C4D1	Combination
SA359427	AG-29	Plasma	Ductal	High BL, High W5	>0	C4D1	Fulvestrant
SA359428	AG-99	Plasma	Ductal	High BL, High W5	>0	C4D1	Fulvestrant
SA359429	AG-73	Plasma	Ductal	High BL, High W5	>0	C4D1	Fulvestrant
SA359430	AG-39	Plasma	Ductal	High BL, Low W5	>0	Baseline	Combination
SA359431	AG-35	Plasma	Ductal	High BL, Low W5	>0	Baseline	Combination
SA359432	AG-106	Plasma	Ductal	High BL, Low W5	>0	Baseline	Combination
SA359433	AG-88	Plasma	Ductal	High BL, Low W5	>0	Baseline	Combination
SA359434	AG-86	Plasma	Ductal	High BL, Low W5	>0	Baseline	Combination
SA359435	AG-13	Plasma	Ductal	High BL, Low W5	>0	Baseline	Fulvestrant
SA359436	AG-60	Plasma	Ductal	High BL, Low W5	>0	Baseline	Fulvestrant
SA359437	AG-56	Plasma	Ductal	High BL, Low W5	>0	Baseline	Fulvestrant
SA359438	AG-48	Plasma	Ductal	High BL, Low W5	>0	Baseline	Fulvestrant
SA359439	AG-11	Plasma	Ductal	High BL, Low W5	>0	Baseline	Fulvestrant
SA359440	AG-90	Plasma	Ductal	High BL, Low W5	>0	Baseline	Fulvestrant
SA359441	AG-87	Plasma	Ductal	High BL, Low W5	>0	C4D1	Combination
SA359442	AG-107	Plasma	Ductal	High BL, Low W5	>0	C4D1	Combination
SA359443	AG-89	Plasma	Ductal	High BL, Low W5	>0	C4D1	Combination
SA359444	AG-40	Plasma	Ductal	High BL, Low W5	>0	C4D1	Combination
SA359445	AG-36	Plasma	Ductal	High BL, Low W5	>0	C4D1	Combination
SA359446	AG-61	Plasma	Ductal	High BL, Low W5	>0	C4D1	Fulvestrant
SA359447	AG-49	Plasma	Ductal	High BL, Low W5	>0	C4D1	Fulvestrant
SA359448	AG-91	Plasma	Ductal	High BL, Low W5	>0	C4D1	Fulvestrant
SA359449	AG-57	Plasma	Ductal	High BL, Low W5	>0	C4D1	Fulvestrant
SA359450	AG-12	Plasma	Ductal	High BL, Low W5	>0	C4D1	Fulvestrant
SA359451	AG-14	Plasma	Ductal	High BL, Low W5	>0	C4D1	Fulvestrant
SA359452	AG-46	Plasma	Ductal	Low BL, Low W5	0	Baseline	Combination
SA359453	AG-74	Plasma	Ductal	Low BL, Low W5	0	Baseline	Combination
SA359454	AG-26	Plasma	Ductal	Low BL, Low W5	0	Baseline	Combination
SA359455	AG-75	Plasma	Ductal	Low BL, Low W5	0	C4D1	Combination
SA359456	AG-27	Plasma	Ductal	Low BL, Low W5	0	C4D1	Combination
SA359457	AG-47	Plasma	Ductal	Low BL, Low W5	0	C4D1	Combination
SA359458	AG-70	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Combination
SA359459	AG-110	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Combination
SA359460	AG-104	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Combination
SA359461	AG-9	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Combination
SA359462	AG-58	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Combination
SA359463	AG-78	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359464	AG-51	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359465	AG-50	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359466	AG-68	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359467	AG-82	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359468	AG-84	Plasma	Ductal	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359469	AG-105	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Combination
SA359470	AG-10	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Combination
SA359471	AG-71	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Combination
SA359472	AG-111	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Combination
SA359473	AG-59	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Combination
SA359474	AG-85	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Fulvestrant
SA359475	AG-79	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Fulvestrant
SA359476	AG-83	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Fulvestrant
SA359477	AG-69	Plasma	Ductal	Low BL, Low W5	>0	C4D1	Fulvestrant
SA359478	AG-52	Plasma	Ductal	not available	0	Baseline	Combination
SA359479	AG-37	Plasma	Ductal	not available	0	Baseline	Combination
SA359480	AG-53	Plasma	Ductal	not available	0	C4D1	Combination
SA359481	AG-38	Plasma	Ductal	not available	0	C4D1	Combination
SA359482	AG-76	Plasma	Ductal	not available	>0	Baseline	Combination
SA359483	AG-18	Plasma	Ductal	not available	>0	Baseline	Combination
SA359484	AG-100	Plasma	Ductal	not available	>0	Baseline	Combination
SA359485	AG-43	Plasma	Ductal	not available	>0	Baseline	Combination
SA359486	AG-20	Plasma	Ductal	not available	>0	Baseline	Combination
SA359487	AG-96	Plasma	Ductal	not available	>0	Baseline	Combination
SA359488	AG-65	Plasma	Ductal	not available	>0	Baseline	Fulvestrant
SA359489	AG-64	Plasma	Ductal	not available	>0	Baseline	Fulvestrant
SA359490	AG-94	Plasma	Ductal	not available	>0	Baseline	Fulvestrant
SA359491	AG-21	Plasma	Ductal	not available	>0	C4D1	Combination
SA359492	AG-44	Plasma	Ductal	not available	>0	C4D1	Combination
SA359493	AG-101	Plasma	Ductal	not available	>0	C4D1	Combination
SA359494	AG-77	Plasma	Ductal	not available	>0	C4D1	Combination
SA359495	AG-97	Plasma	Ductal	not available	>0	C4D1	Combination
SA359496	AG-19	Plasma	Ductal	not available	>0	C4D1	Combination
SA359497	AG-66	Plasma	Ductal	not available	>0	C4D1	Fulvestrant
SA359498	AG-95	Plasma	Ductal	not available	>0	C4D1	Fulvestrant
SA359499	AG-108	Plasma	Ductal	not available	>0	C4D1	Fulvestrant
SA359500	AG-7	Plasma	Lobular	High BL, High W5	>0	Baseline	Combination
SA359501	AG-8	Plasma	Lobular	High BL, High W5	>0	C4D1	Combination
SA359502	AG-1	Plasma	Lobular	High BL, Low W5	0	Baseline	Combination
SA359503	AG-2	Plasma	Lobular	High BL, Low W5	0	C4D1	Combination
SA359504	AG-3	Plasma	Lobular	Low BL, Low W5	0	Baseline	Fulvestrant
SA359505	AG-5	Plasma	Lobular	Low BL, Low W5	0	Baseline	Fulvestrant
SA359506	AG-4	Plasma	Lobular	Low BL, Low W5	0	C4D1	Fulvestrant
SA359507	AG-6	Plasma	Lobular	Low BL, Low W5	0	C4D1	Fulvestrant
SA359508	AG-15	Plasma	Lobular	Low BL, Low W5	>0	Baseline	Combination
SA359509	AG-41	Plasma	Lobular	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359510	AG-24	Plasma	Lobular	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359511	AG-92	Plasma	Lobular	Low BL, Low W5	>0	Baseline	Fulvestrant
SA359512	AG-16	Plasma	Lobular	Low BL, Low W5	>0	C4D1	Combination
SA359513	AG-42	Plasma	Lobular	Low BL, Low W5	>0	C4D1	Fulvestrant
SA359514	AG-93	Plasma	Lobular	Low BL, Low W5	>0	C4D1	Fulvestrant
SA359515	AG-25	Plasma	Lobular	Low BL, Low W5	>0	C4D1	Fulvestrant
SA359516	AG-22	Plasma	Lobular	not available	0	Baseline	Combination
SA359517	AG-23	Plasma	Lobular	not available	0	C4D1	Combination

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Collection:

Collection ID:	CO003429
Collection Summary:	Lithium-heparin (LiHep) plasma samples were obtained every 4 weeks until surgery and one month after surgery. After collection LiHep vacutainers were centrifuged for 20 minutes (600 x g), separated plasma was transferred to a 15 mL conical tube, centrifuged a second time for 15 minutes (1500 x g), aliquoted into 500 uL aliquots, and stored at -80⁰C until analysis.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR003445
Treatment Summary:	Study design and treatments NCT02955394 (COMIRB 16-1042) was an open-label randomized phase II trial of fulvestrant with or without enzalutamide. Fulvestrant 500 mg IM was administered on day 1, 15, 29, and then every 4 weeks for a total of 4 months. Enzalutamide 160 mg po daily was given concurrently for 4 months for those women assigned to the combination (Fig. 1A). Surgery was anticipated to occur immediately after week 17, upon completion of enzalutamide; however timing was subject to surgeon and operating suite availability. As drug supply was limited, enzalutamide could not always be continued until the day prior to surgery as originally planned. Stratification factors were institution, clinical node status (N0,N+), and T-stage (T2,T3/4). Pre- or peri-menopausal women received concurrent ovarian suppression with a gonadotropinreleasing hormone agonist. The study was conducted at the Universities of Colorado and Tennessee and Memorial Sloan Kettering Cancer Center. The study protocol and its amendments 16 were approved by the respective Institutional Review Boards as well as the Department of Defence HRPO committee. All patients provided written informed consent prior to participating in the study. The study was conducted under the principles of the World Medical Association, Declaration of Helsinki, and Good Clinical Practice guidelines of the International Conference on Harmonisation. The study did not require an Investigational New Drug Application. Drug support (enzalutamide) was provided by Astellas and Pfizer as part of this investigator-sponsored research study. Study population Eligible patients were women ≥18 years of age with adequate organ and bone marrow function and an ECOG performance score (PS) of 2 or less. All had primary breast cancer of at least 2 cm in size (>T2, N0-2, M0) determined to be ER positive and HER2 negative. Men were excluded due to potential confounding from androgenic stimuli. History of seizures was exclusionary due to the toxicity profile of enzalutamide. Determination of AR expression was not a requirement as it was expected that ~90% of tumors would stain for AR, and the assay has not yet been validated for clinical decision-making. Concomitant medications with substantial pharmacokinetic (PK) interaction with enzalutamide were avoided. Safety and antitumor assessment All patients who received at least one dose of enzalutamide were assessed for safety biweekly for the first 4 weeks, then every 4 weeks until 30 days after the last dose of enzalutamide. Safety and tolerability were determined by assessment of adverse events (AEs), physical examinations, ECOG PS, vital signs, and laboratory tests. The severity of abnormal laboratory values and AEs 17 were classified using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 4.03. SAEs were also evaluated by Astellas Pharma Global Development – United States. A monthly teleconference was held amongst the institutional investigators to review patients and adverse events. An institutional Data Safety and Monitoring Committee at University of Colorado also had oversight for monitoring. Tissue acquisition Fresh tumor biopsies (core needle biopsies of the breast) were required at study entry (BL), and after 4 weeks on therapy (when both Fulv and E were likely at steady state concentrations) and these were termed “W5” biopsy. Fresh frozen and fixed tissue was collected at the time of surgery.

Treatment ID:

TR003445

Treatment Summary:

Study design and treatments NCT02955394 (COMIRB 16-1042) was an open-label randomized phase II trial of fulvestrant with or without enzalutamide. Fulvestrant 500 mg IM was administered on day 1, 15, 29, and then every 4 weeks for a total of 4 months. Enzalutamide 160 mg po daily was given concurrently for 4 months for those women assigned to the combination (Fig. 1A). Surgery was anticipated to occur immediately after week 17, upon completion of enzalutamide; however timing was subject to surgeon and operating suite availability. As drug supply was limited, enzalutamide could not always be continued until the day prior to surgery as originally planned. Stratification factors were institution, clinical node status (N0,N+), and T-stage (T2,T3/4). Pre- or peri-menopausal women received concurrent ovarian suppression with a gonadotropinreleasing hormone agonist. The study was conducted at the Universities of Colorado and Tennessee and Memorial Sloan Kettering Cancer Center. The study protocol and its amendments 16 were approved by the respective Institutional Review Boards as well as the Department of Defence HRPO committee. All patients provided written informed consent prior to participating in the study. The study was conducted under the principles of the World Medical Association, Declaration of Helsinki, and Good Clinical Practice guidelines of the International Conference on Harmonisation. The study did not require an Investigational New Drug Application. Drug support (enzalutamide) was provided by Astellas and Pfizer as part of this investigator-sponsored research study. Study population Eligible patients were women ≥18 years of age with adequate organ and bone marrow function and an ECOG performance score (PS) of 2 or less. All had primary breast cancer of at least 2 cm in size (>T2, N0-2, M0) determined to be ER positive and HER2 negative. Men were excluded due to potential confounding from androgenic stimuli. History of seizures was exclusionary due to the toxicity profile of enzalutamide. Determination of AR expression was not a requirement as it was expected that ~90% of tumors would stain for AR, and the assay has not yet been validated for clinical decision-making. Concomitant medications with substantial pharmacokinetic (PK) interaction with enzalutamide were avoided. Safety and antitumor assessment All patients who received at least one dose of enzalutamide were assessed for safety biweekly for the first 4 weeks, then every 4 weeks until 30 days after the last dose of enzalutamide. Safety and tolerability were determined by assessment of adverse events (AEs), physical examinations, ECOG PS, vital signs, and laboratory tests. The severity of abnormal laboratory values and AEs 17 were classified using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 4.03. SAEs were also evaluated by Astellas Pharma Global Development – United States. A monthly teleconference was held amongst the institutional investigators to review patients and adverse events. An institutional Data Safety and Monitoring Committee at University of Colorado also had oversight for monitoring. Tissue acquisition Fresh tumor biopsies (core needle biopsies of the breast) were required at study entry (BL), and after 4 weeks on therapy (when both Fulv and E were likely at steady state concentrations) and these were termed “W5” biopsy. Fresh frozen and fixed tissue was collected at the time of surgery.

Sample Preparation:

Sampleprep ID:	SP003443
Sampleprep Summary:	Plasma aliquots were thawed on ice then metabolites extracted from a 20 uL plasma aliquot using 480 uL of cold 5:3:2 MeOH:acetonitrile:water. Samples were vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C) and transferred to autosampler vials.
Processing Storage Conditions:	4℃
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN005428	AN005429
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Orbitrap Exploris 120	Thermo Orbitrap Exploris 120
Ion Mode	NEGATIVE	POSITIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH004117
Chromatography Summary:	Negative C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B
Flow Rate:	450 uL/min
Sample Injection:	10 uL
Solvent A:	95% water/5% acetonitrile; 1 mM ammonium acetate
Solvent B:	95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:	Reversed phase

Chromatography ID:	CH004118
Chromatography Summary:	Positive C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B
Flow Rate:	450 uL/min
Sample Injection:	10 uL
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005154
Analysis ID:	AN005428
Instrument Name:	Thermo Orbitrap Exploris 120
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) detection duration 20 msec, source voltage 2.0 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:	NEGATIVE

MS ID:	MS005155
Analysis ID:	AN005429
Instrument Name:	Thermo Orbitrap Exploris 120
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) detection duration 20 msec, source voltage 3.5 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:	POSITIVE