Window Trial of Abemaciclib for Surgically Resectable, Chemotherapy-Resistant, Triple Negative Breast Cancer (a BEAUTY Study*)
Phase: PHASE2
Conditions: Anatomic Stage I Breast Cancer AJCC v8, Anatomic Stage II Breast Cancer AJCC v8, Anatomic Stage III Breast Cancer AJCC v8, Breast Ductal Carcinoma In Situ, Breast Fibrocystic Change, Breast Lobular Carcinoma In Situ, Invasive Breast Carcinoma, Prognostic Stage I Breast Cancer AJCC v8, Prognostic Stage IA Breast Cancer AJCC v8, Prognostic Stage IB Breast Cancer AJCC v8, Prognostic Stage II Breast Cancer AJCC v8, Prognostic Stage IIA Breast Cancer AJCC v8, Prognostic Stage IIB Breast Cancer AJCC v8, Prognostic Stage III Breast Cancer AJCC v8, Prognostic Stage IIIA Breast Cancer AJCC v8, Prognostic Stage IIIB Breast Cancer AJCC v8, Prognostic Stage IIIC Breast Cancer AJCC v8, Triple-Negative Breast Carcinoma
Sponsor: Mayo Clinic

 
 
 
 
 
MC1734 / 17-010660 
 
 
Window Trial of Abemaciclib for Surgically Resectable, 
Chemotherapy-Resistant, Triple Negative Breast Cancer  
(a BEAUTY Study*) 
 
 
 
NCT03979508 
 
 
Document Date: 04/11/2024 
 
 
 
*Breast Cancer Genome-Guided Therapy Study 

 
 
 
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MC1734 Window Trial of Abemaciclib for Surgically Resectable, Chemotherapy-Resistant, 
Triple Negative Breast Cancer (a BEAUTY study*) 
*Breast Cancer Genome-Guided Therapy Study 
Protocol Number 
Mayo Clinic Protocol Number: MC1734 
Protocol Chairs 
Matthew P Goetz MD / Judy C Boughey MD 
Mayo Clinic 
200 Second St SW 
Rochester MN 55905 
Phone: 507/284-2511 
Email: goetz.matthew@mayo.edu / boughey.judy@mayo.edu  
Co-Investigators 
Pooja Advani MD  
Sarah A McLaughlin MD  
Brenda Ernst MD 
Barbara A Pockaj MD  
Malvika H Solanki MBBS PhD 
Ann E McCullough MD 
Aziza Nassar MD MPH 
Katie N Hunt MD 
Bhavika Patel MD 
Kristin A Robinson MD 
Krishna R Kalari PhD  
Liewei Wang MD PhD  
Zhenkun Lou PhD 
Roberto A Leon Ferre MD 
Jose C (JC) Villasboas Bisneto MD 
Statistician 
Vera J Suman PhD 
Mayo Clinic 
Rochester MN 
Supporters 
Eli Lilly and Company 
 

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Window Trial of Abemaciclib for Surgically Resectable, Chemotherapy Resistant Triple 
Negative Breast Cancer 
Study Reference Numbers 
Organization 
Reference number 
Mayo Clinic 
MC1734 (IRB 17-010660) 
Eli Lilly 
I3Y-US-I010 
ClinicalTrials.gov 
NCT03979508 
FDA 
IND 142085 
 
 
 
 
 
- Protocol Revision Record - 
Original Protocol: 
16Apr2019  
Activation to Mayo Clinic 
10Jan2020 
MCCC Amendment 1 
16Jun2020 
MCCC Amendment 2 
10Dec2020 
MCCC Amendment 3 
03Mar2021 
MCCC Amendment 4 
04Aug2021 
MCCC Amendment 5 
04Mar2022 
MCCC Amendment 6 
09Jun2022 
MCCC Amendment 7 
11Apr2024 
 
 
 
 
 

Window trial Abemaciclib TNBC 
 
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MC1734 
 
 
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TABLE OF CONTENTS 
TABLE OF CONTENTS ............................................................................................................................................ 3 
SCHEMA ...................................................................................................................................................................... 6 
1. 
STUDY DESIGN/SUMMARY ............................................................................................................................ 7 
2. 
OBJECTIVES ....................................................................................................................................................... 8 
2.1 
PRIMARY OBJECTIVE .................................................................................................................................... 8 
2.2 
SECONDARY OBJECTIVES FOR COHORT A AND COHORT B INDEPENDENTLY ................................................ 8 
2.3 
EXPLORATORY OBJECTIVES FOR COHORT A AND COHORT B INDEPENDENTLY ............................................ 9 
3. 
BACKGROUND ................................................................................................................................................... 9 
3.1 
TRIPLE NEGATIVE BREAST CANCER ............................................................................................................. 9 
3.2 
TNBC SUBTYPES ........................................................................................................................................ 10 
3.3 
PRECLINICAL ACTIVITY OF CDK 4/6 INHIBITORS IN ER NEGATIVE BREAST CANCER .................................. 10 
3.4 
CLINICAL IMPORTANCE OF IMMUNE AND EMT BIOMARKERS IN TNBC. ..................................................... 13 
3.5 
MAYO CLINIC PROSPECTIVE NEOADJUVANT CHEMOTHERAPY STUDY ....................................................... 14 
3.6 
ABEMACICLIB (VERZENIO®) ...................................................................................................................... 14 
3.7 
ASSESSING RESPONSE TO NEOADJUVANT CHEMOTHERAPY ......................................................................... 17 
3.8 
TRIAL SUMMARY ........................................................................................................................................ 18 
3.9 
LOWERING OF STARTING DOSE OF ABEMACICLIB ....................................................................................... 18 
3.10 
ADDITION OF COHORT B (EVALUATING EFFECTS OF ABEMACICLIB IN TNBC PATIENTS TREATED WITH 
PEMBROLIZUMAB AND NAC) ..................................................................................................................... 19 
4. 
PARTICIPANT SELECTION .......................................................................................................................... 20 
4.1 
PRE-REGISTRATION INCLUSION CRITERIA .................................................................................................. 20 
4.2 
PRE-REGISTRATION EXCLUSION CRITERIA ................................................................................................. 21 
4.3 
REGISTRATION - INCLUSION CRITERIA FOR ALL PATIENTS ......................................................................... 22 
4.4 
REGISTRATION - EXCLUSION CRITERIA FOR COHORT B GROUP 4 ONLY ..................................................... 23 
4.5 
INCLUSION OF UNDERREPRESENTED POPULATIONS .................................................................................... 23 
5. 
REGISTRATION PROCEDURES ................................................................................................................... 24 
5.1 
GROUPING FACTORS FOR ELIGIBLITY CHECKLIST ...................................................................................... 24 
5.2 
PRE-REGISTRATION (STEP 0) ...................................................................................................................... 24 
5.3 
REGISTRATION PROCEDURES ...................................................................................................................... 25 
6. 
STUDY CALENDAR ......................................................................................................................................... 27 
6.1 
COHORT B GROUP 3 STUDY CALENDAR – NO INVASIVE DISEASE ............................................................. 27 
6.2 
COHORT B GROUP 4 STUDY CALENDAR – RESIDUAL INVASIVE DISEASE .................................................. 28 
7. 
TREATMENT PLAN ........................................................................................................................................ 30 
7.1 
BIOPSY DURING PREREGISTRATION PHASE .................................................................................................. 30 
7.2 
COHORT B GROUP 4: ABEMACICLIB ADMINISTRATION .............................................................................. 30 
7.3 
COHORT B GROUP 4 CONCOMITANT TREATMENT AND SUPPORTIVE CARE GUIDELINES ............................ 31 
7.4 
DURATION OF THERAPY .............................................................................................................................. 31 
7.5 
DURATION OF FOLLOW-UP ......................................................................................................................... 32 
7.6 
CRITERIA FOR STUDY DISCONTINUATION ................................................................................................... 32 
7.7 
TREATMENT FOLLOWING SURGERY ............................................................................................................ 32 
8. 
EXPECTED ADVERSE EVENTS AND DOSING DELAYS/DOSE MODIFICATIONS –COHORT B 
GROUP 4 ............................................................................................................................................................ 33 

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8.1 
TREATMENT ADMINISTRATION ................................................................................................................... 33 
8.2 
ANTICIPATED ADVERSE EVENTS ................................................................................................................ 33 
8.3 
MANAGEMENT OF ADVERSE EVENTS .......................................................................................................... 34 
8.4 
DOSE MODIFICATIONS/ DELAYS ................................................................................................................. 34 
9. 
DRUG FORMULATION/STORAGE/SUPPLY ............................................................................................. 38 
9.1 
ABEMACLICLIB (VERZENIO®, LY2835219, NSC 783671) .......................................................................... 38 
10. CORRELATIVE/SPECIAL STUDIES ............................................................................................................ 40 
10.1 
COLLECTION TABLES .................................................................................................................................. 40 
10.2 
BLOOD SAMPLES ........................................................................................................................................ 42 
10.3 
STOOL SAMPLES ......................................................................................................................................... 43 
10.4 
TISSUE SAMPLES ......................................................................................................................................... 44 
10.5 
GENETIC TESTING ....................................................................................................................................... 47 
10.6 
ADDITIONAL INFORMATION ........................................................................................................................ 47 
11. SPECIMEN BANKING ..................................................................................................................................... 47 
12. MEASUREMENT OF EFFECT: COHORT A GROUP 2 AND COHORT B GROUP 4 ........................... 47 
13. ADVERSE EVENT REPORTING REQUIREMENTS .................................................................................. 48 
13.1 
GENERAL .................................................................................................................................................... 48 
13.2 
DEFINITIONS ............................................................................................................................................... 48 
13.3 
REPORTING PROCEDURES ........................................................................................................................... 49 
14. DATA MONITORING ...................................................................................................................................... 57 
14.1 
MONITORING .............................................................................................................................................. 57 
15. REGULATORY CONSIDERATIONS ............................................................................................................ 58 
15.1 
PROTOCOL REVIEW AND AMENDMENTS ..................................................................................................... 58 
15.2 
INFORMED CONSENT ................................................................................................................................... 58 
15.3 
ETHICS AND GCP ........................................................................................................................................ 58 
15.4 
COMPLIANCE WITH TRIAL REGISTRATION AND RESULTS POSTING REQUIREMENTS ................................... 59 
16. STUDY CONDUCT GUIDELINES ................................................................................................................. 59 
16.1 
STUDY DOCUMENTATION ........................................................................................................................... 59 
16.2 
RECORDS RETENTION ................................................................................................................................. 59 
16.3 
PUBLICATION .............................................................................................................................................. 59 
17. STATISTICAL CONSIDERATIONS .............................................................................................................. 60 
17.1 
BACKGROUND ............................................................................................................................................. 60 
17.2 
STUDY DESIGN ........................................................................................................................................... 60 
17.3 
SECONDARY ENDPOINTS AND ANALYSIS PLANS FOR EACH COHORT INDEPENDENTLY ................................ 61 
17.4 
CORRELATIVE ENDPOINTS FOR EACH COHORT INDEPENDENTLY ................................................................. 62 
17.5 
EXPLORATORY OBJECTIVES FOR EACH COHORT INDEPENDENTLY ............................................................... 62 
17.6 
ACCRUAL AND STUDY DURATION ............................................................................................................... 63 
17.7 
SAFETY MONITORING .................................................................................................................................. 64 
17.8 
INCLUSION OF WOMEN AND MINORITIES .................................................................................................... 64 
18. BUDGET ............................................................................................................................................................. 65 
18.1 
COSTS CHARGED TO PATIENT ...................................................................................................................... 65 
18.2 
TREATMENT AND TESTS TO BE RESEARCH FUNDED ..................................................................................... 65 

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19. REFERENCES ................................................................................................................................................... 66 
19.1 
ADDITIONAL REFERENCES FOR TRANSLATIONAL STUDIES ......................................................................... 69 
APPENDICES ............................................................................................................................................................ 70 
APPENDIX A: ECOG-KARNOFSKY PERFORMANCE STATUS SCALE ...................................................................... 71 
APPENDIX B: STUDY DRUG DIARY (GROUP 4 ONLY) ........................................................................................... 72 
APPENDIX C: LILLY GUIDANCE FOR HEPATIC TREATMENT EMERGENT ABNORMALITY ...................................... 73 

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SCHEMA 
COHORT A: Patients treated with a neoadjuvant chemotherapy regimen without 
pembrolizumab – Closed with MCCC Amendment 6 
COHORT B: Patients treated with a neoadjuvant chemotherapy regimen in 
combination with pembrolizumab 
 
 
 Pre-Registration  
 
 
 
 
 
 
 
 
 
 
 
 
 
Biopsy 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Registration 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cohort A Group 1 closed to enrollment 
Cohort B Group 3: No invasive disease 
on H&E 
 
 
 
 
Cohort A Group 2 closed to enrollment 
Cohort B Group 4: Invasive disease on 
H&E 
 
 
 
 
 
 
 
 
Surgery 
 
 
 
 
Abemaciclib 14-21 days 
such that last planned dose is taken 
on the day prior to surgery* 
 
 
 
 
 
 
 
 
Collection of surgical and pathology 
reports 
 
 
 
 
Surgery** 
 
 
 
 
 
 
 
 
Off study 
 
 
 
 Collection of surgical tissue, and surgical 
and pathology reports  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Follow-up 30-60 days post-surgery 
(End of Study visit) 
 
 
 
 
 
 
 
 
 
 
 
 
 
Off study 
* If abemaciclib is administered for 14-21 days (or up to 42 total doses) and surgery is performed 
more than 3 days after the last dose of abemaciclib surgical tissue should still be submitted 
**Or research biopsy if surgery will not be done 
Cycle = 14-21 days of treatment (up to 42 total doses);  
NOTE: Patient will not be allowed to register if >56 days after completion of neoadjuvant therapy. 
Generic name: abemaciclib 
Brand name(s): Verzenio® 
Availability: Eli Lilly & Co supplied 

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1. STUDY DESIGN/SUMMARY 
Triple negative breast cancer (TNBC), defined by a lack of estrogen receptor (ER) alpha and 
progesterone receptor (PR) expression and absence of HER2 amplification, is an aggressive 
and highly metastatic subset. Although neoadjuvant chemotherapy combined with 
pembrolizumab reduces recurrence and death [ref: PMID: 32101663], chemotherapy 
resistance remains a major problem. Patients with residual disease after neoadjuvant-based 
chemotherapy (NAC) have a distant recurrence risk approaching 50%.1 
At least four well-characterized molecular subtypes of TNBC exist, including basal, 
mesenchymal, immune-enriched and luminal androgen receptor (LAR) subtypes.2 These 
subtypes respond differently to NAC. For example, a higher proportion of women with the 
LAR subset of TNBC are resistant to NAC and exhibit residual disease compared to non-
LAR (e.g. basal) subsets.3,4 However, it is not known whether the survival benefit of 
chemotherapy differs amongst these TNBC subsets. 
Palbociclib, ribociclib and abemaciclib are cyclin dependent kinase 4 and 6 inhibitors (CDK 
4/6i) that have recently been FDA approved for the treatment of ER positive, metastatic 
breast cancer (MBC), either alone (abemaciclib)5 or in combination with endocrine therapy 
(palbociclib,6,7 ribociclib,8 and abemaciclib).9,10 Estrogen receptor (ER) and Rb status are 
thought to be critical determinants of CDK4/6i antitumor activity.11 
Recently, there have been a series of pre-clinical and clinical publications documenting 
novel aspects by which CDK 4/6i promote anti-tumor activity, including in TNBC: 
1) Activation of the immune system: Goel et al. identified that CDK 4/6 inhibition 
promotes antitumor immunity through activation of tumor cell expression of endogenous 
retroviral elements and intracellular levels of double-stranded RNA, resulting in the 
production of type III interferons and tumor antigen presentation.12 Additionally, Goel et 
al demonstrated that CDK4/6i suppress the proliferation of regulatory T cells and DNA 
methyltransferase 1, resulting in cytotoxic T-cell-mediated clearance of tumor cells. In 
this study, using the MMTV-rtTA/tetO-HER2 tumour model, the combination of 
abemaciclib plus an anti-PDL1 antibody regressed tumors to a greater degree than either 
abemaciclib alone or anti-PDL1 alonethe combination of abemaciclib plus an anti-PDL1 
antibody regressed to a greater degree than either abemaciclib alone or anti-PDL1 alone. 
This study demonstrated that the CDK4/6 inhibitor abemaciclib may enhance anti-
tumour immunity by increasing the functional capacity of tumour cells to present 
antigen as well as by reducing the immunosuppressive TReg population. 
 
Based on the preclinical data outlined above, a phase I study was performed, combining 
abemaciclib plus pembrolizumab plus anastrozole in post-menopausal HR+, HER2- pts 
with locally advanced or mBC. Patients received 150-mg abemaciclib orally every 
12 hours plus pembrolizumab 200 mg intravenously on Day 1 every 21 days plus 
anastrozole 1 mg orally every 24 hours. Grade 3/4 AEs included increased alanine 
aminotransferase and neutropenia (8 pts each, 31%) and increased aspartate 
aminotransferase (6 pts, 23%) and 2 Grade 5 events related to pneumonitis. Preliminary 
response assessment demonstrated that 5 patients had a confirmed partial response (19% 
ORR) and clinical benefit rate (CR+PR+SD persisting for ≥6 months) was 27%. 

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Therefore, despite strong preclinical rationale, further development of the combination 
of abemaciclib and pembrolizumab is on hold based on these toxicity data. 
2) Effects on epithelial-mesenchymal transition and inhibition of metastases. Epithelial-
mesenchymal transition (EMT) is a highly-conserved cellular program in which 
polarized, immobile epithelial cells are converted to migratory mesenchymal cells, a 
critical process that occurs during embryogenesis,13,14 and important for tissue 
regeneration, tumor progression, metastasis, and chemoresistance.14-22 Liu et al23 
identified a novel mechanism by which CDK4/6i alter EMT and inhibit metastases in 
TNBC. They demonstrated that overexpression of DUB3 increased SNAIL levels and 
conversely, knockdown of DUB3 decreased SNAIL and vimentin protein levels, without 
affecting mRNA levels. CDK4/6 phosphorylates DUB3 at Ser41, thus activating DUB3. 
Treatment of cells with palbociclib inhibited DUB3 activity, decreased SNAIL and 
vimentin expression and decreased cell migration. These findings were confirmed with 
the drug abemaciclib. 
For patients with TNBC treated with NAC with/without pembrolizumab and with residual 
disease, our specific hypotheses are that abemaciclib treatment for 14-21 days will: 
1) Increase CD8 T cells and decrease FOXP3 regulatory cells (i.e., increase CD8/FOX3 
ratio) 
2) Decrease vimentin expression 
3) Increase expression of antigen presentation-associated genes in tumors with 
functional Rb 
4) Inhibit proliferation (Ki-67) within subsets with functional Rb (e.g., LAR)  
2. OBJECTIVES 
2.1 Primary Objective 
(1) To examine the effects of abemaciclib on the CD8/FOXP3 ratio in chemotherapy 
resistant TNBC patients following a neoadjuvant chemotherapy regimen without the 
addition of pembrolizumab (Cohort A) 
(2) To examine the effects of abemaciclib on the CD8/FOXP3 ratio in chemotherapy 
resistant TNBC patients following a neoadjuvant chemotherapy regimen with the 
addition of pembrolizumab (Cohort B) 
CD8/FOXP3 protein expression will be determined in tumor tissue obtained by image 
guided percutaneous breast biopsy after completion of NAC+/- pembrolizumab (prior to 
abemaciclib) and in residual invasive breast cancer obtained at surgery (after 14-21 days 
of abemaciclib).  
2.2 Secondary Objectives for Cohort A and Cohort B independently 
1) Assess abemaciclib toxicities 
2) To examine the effects of abemaciclib on the percentage of vimentin expressing 
invasive cancer cells 
3) Within TNBC molecular subtypes (basal, mesenchymal, and LAR), to evaluate the 
effects of abemaciclib on: 

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a) the individual elements of tumor grade (mitoses, nuclear pleomorphism, and 
tubule formation) 
b) tumor proliferation (as measured by tumor Ki-67 and serum TKI) 
c) pDUB3 as well as EMT markers including SNAIL/SLUG, TWIST, and E-
Cadherin as measured by IHC 
d) Quantification of tumor-infiltrating lymphocytes (as examined by H&E) 
2.3 Exploratory Objectives for Cohort A and Cohort B independently 
1) To evaluate the effect of abemaciclib on tumor RNA expression.  
2) To evaluate the effects of abemaciclib on the immune phenotype of peripheral blood 
mononuclear cells (PBMC), by evaluating expression of a panel of cell surface 
markers optimized of identification of human immune cell subpopulations. This goal 
will be accomplished using multiplexed single-cell proteomic technologies (e.g. 
Helios CyTOF platform). 
3) To evaluate the effects of abemaciclib on tumor-infiltrating immune cells in formalin-
fixed paraffin-embedded (FFPE) tumor sections, using multiplexed imaging 
technologies (e.g imaging mass cytometry , Nanostring DSP or CODEX) which will 
include: 
a) genes directly involved in tumor cell antigen presentation (e.g. B2M, HLA-A, 
HLA-B, HLA-C, TAP1, TAP2, TAPBP) 
b) interferon-stimulated genes (ISGs) that regulate antigen presentation (e.g. STAT1, 
NLRC5) and other ISGs (e.g. IRFs, OAS2) 
c) genes involved in dsRNA response (e.g. DDX58, DHX58) 
d) genes encoding interferons, including type 3 IFNs (e.g. IFNL1, IFNL2, IFNL3) 
e) genes indicating a cytotoxic T cell response (e.g. PRF1, GZMB) 
f) Treg-specific transcription factor genes (e.g. FOXP3, IKZF2)  
4) To assess the difference in the frequency of JAK-2 amplification among patients 
whose post-abemaciclib CD8/FOXP3 ratio ≥1.6 and that among patients whose post-
abemaciclib CD8/FOXP3 ratio <1.6. 
5) To generate organoids for future research. 
6) To evaluate changes in the microbiome with exposure to abemaciclib. 
3. BACKGROUND 
3.1 Triple Negative Breast Cancer 
Triple negative breast cancer (TNBC), defined by a lack of ERα and PR expression and 
absence of HER2 amplification, accounts for up to 20% of all breast cancers. This 
subtype is clinically unique due to its aggressive and metastatic nature, typically 
presenting in younger women at an advanced stage. Despite extensive efforts to 
characterize the molecular drivers associated with TNBC, including large scale genomic 
and proteogenomic sequencing efforts24-26, standard treatment recommendations (surgery, 
chemotherapy and radiation) have not changed substantially over the past 20 years. 
Systemic chemotherapy is administered in the adjuvant setting (after definitive breast 
surgery) or neoadjuvant setting, with multiple studies demonstrating that chemotherapy 

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reduces recurrence and prolongs survival.27 Chemotherapy is standardly administered in 
the neoadjuvant setting (prior to surgery) for TNBC. A substantial benefit of this 
approach is the prognostic information gained from pathological assessment of the tumor 
bed and lymph nodes after surgery, reflecting the response/resistance to neoadjuvant 
chemotherapy (NAC). Complete eradiation of invasive cancer within the breast and 
nodes, called pathological complete response (pCR), is a strong prognostic factor.1 
Although anthracycline and taxane based chemotherapy substantially reduces the risk of 
recurrence and death, chemotherapy resistance is a major problem. Patients with residual 
disease after neoadjuvant chemotherapy (no pCR) have a distant recurrence risk 
approaching 50.1 For these reasons, novel therapeutic options, including new 
biomarkers and drug targets, are greatly needed for TNBC patients with resistant 
disease.  
3.2 TNBC Subtypes 
At least four well characterized molecular subtypes of TNBC exist, including basal, 
mesenchymal, immune-enriched and LAR subtypes.2 These subtypes respond differently 
to NAC. For example, a higher proportion of women with the LAR subset of TNBC 
treated with NAC exhibit residual disease compared to non-LAR (e.g. basal) subsets.3,4 
However, it is not known whether the survival benefit of chemotherapy differs amongst 
these TNBC subsets. 
Palbociclib, ribociclib and abemaciclib are cyclin dependent kinase 4 and 6 inhibitors 
(CDK 4/6i) are FDA approved for the treatment of ER positive, metastatic breast cancer 
(MBC), either alone (abemaciclib)5 or in combination with endocrine therapy 
(palbociclib,6,7 ribociclib,8 and abemaciclib9,10). Estrogen receptor (ER) and Rb status are 
thought to be critical determinants of CDK4/6i antitumor activity.11 
3.3 Preclinical activity of CDK 4/6 inhibitors in ER negative breast cancer 
Recently, there have been a series of pre-clinical and clinical publications documenting 
novel aspects by which CDK 4/6i promote anti-tumor activity, including in TNBC: 
 Activation of the immune system 
Goel et al. identified that CDK 4/6 inhibition promotes antitumor immunity through 
activation of tumor cell expression of endogenous retroviral elements and 
intracellular levels of double-stranded RNA, resulting in the production of type III 
interferons and tumor antigen presentation.12 Additionally, Goel et al demonstrated 
that CDK4/6i suppress the proliferation of regulatory T cells and DNA 
methyltransferase 1, resulting in cytotoxic T-cell-mediated clearance of tumor cells. 
Goel et al demonstrated that in tumor free mice, both abemaciclib and palbociclib 
significantly reduced Treg numbers and the Treg:CD8 ratio in the spleen and lymph 
nodes, demonstrating tumor-independent effects of these agents. The selective 
suppression of Treg proliferation (and not that of CD8+ or naïve CD4+ T cells) may 
relate to the fact that Tregs express higher levels of Rb1, a key mediator of CDK4/6 
pathway modulation. Notably, Rb1 expression is over 3-fold higher in Tregs than 
CD8+ T cells. 

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To determine whether cytotoxic T lymphocytes (CTLs) were involved in 
abemaciclib-mediated therapeutic efficacy, Goel et al implanted MMTV-rtTA/tetO-
HER2 tumor fragments orthotopically into athymic Foxn1nu mice maintained on 
doxycycline, and treated them once tumors were established. In contrast to tumors in 
immunocompetent mice, abemaciclib-treated tumors in nude mice continued to 
grow. Furthermore, in tumor-bearing MMTV-rtTA/tetO-HER2 mice treated with an 
anti-CD8 antibody prior to abemaciclib administration, tumor regression (observed 
in control IgG treated mice), was significantly mitigated by CD8 neutralization. 
Hence, tumor regression mediated by CDK4/6 inhibition is, at least in part, 
dependent on the presence of CTLs.  
Finally, using the MMTV-rtTA/tetO-HER2 tumour model, Goel et al demonstrated 
that the combination of abemaciclib plus an anti-PDL1 antibody regressed tumors to 
a greater degree than either abemaciclib alone or anti-PDL1 alone. 
Following the publication of Goel et al, others have validated these findings and 
observed a robust effect of abemaciclib on antigen presentation pathway in Rb-
competent breast cancer cells, which are further potentiated in vivo, leading to 
changes in major histocompatibility complex (MHC) class I and II expression on 
tumor cells, and antigen-presenting cells. Specifically, Schaer et al demonstrated 
that abemaciclib treatment modulates antigen presentation mechanisms (e.g., 
upregulation of MHC class I genes HLA-A, HLA-C, HLA-G, transporter associated 
with antigen processing TAP-1, immunoproteasome subunit LMP-7 encoded by 
PSMB8) in tumor cells in vitro, triggering an immune response and “activation” of 
cytotoxic CD8+ T cells.28 
Based on these preclinical data, a phase I study was performed, combining 
abemaciclib plus pembrolizumab plus anastrozole in post-menopausal HR+, HER2- 
pts with locally advanced or mBC. Patients received 150-mg abemaciclib orally 
every 12 hours plus pembrolizumab 200 mg intravenously on Day 1 every 21 days 
plus anastrozole 1 mg orally every 24 hours. Grade 3/4 AEs included increased 
alanine aminotransferase and neutropenia (8 pts each, 31%) and increased aspartate 
aminotransferase (6 pts, 23%) and two Grade 5 events related to pneumonitis. 
Preliminary response assessment demonstrated that 5 patients had a confirmed 
partial response (19% ORR) and clinical benefit rate (CR+PR+SD persisting for 
≥6 months) was 27%. Therefore, despite strong preclinical rationale further 
development of the combination of abemaciclib and pembrolizumab is on hold 
based on these toxicity data. 
 Effects on epithelial-mesenchymal transition and inhibition of metastases 
Epithelial-mesenchymal transition (EMT) is a highly-conserved cellular program in 
which polarized, immobile epithelial cells are converted to migratory mesenchymal 
cells, a critical process that occurs during embryogenesis,14,15 and important for 
tissue regeneration, tumor progression, metastasis, and chemoresistance.15-22,29 Loss 
of cell adhesion molecule E-cadherin is a functional event of EMT, altering the 
phenotype of epithelial cells from noninvasive to invasive.18,30 SNAIL directly 
represses CDH, the gene encoding E-cadherin, and activates the expression of 
invasion-associated genes, thereby promoting EMT.23,31,32  

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Liu et al23 identified a novel mechanism by which CDK4/6i alter EMT and inhibit 
metastases in TNBC. They demonstrated that overexpression of DUB3 increased 
SNAIL levels and conversely, knockdown of DUB3 decreased SNAIL and 
vimentin protein levels, without affecting mRNA levels. While DUB3 had no 
apparent effect on cell growth, knockdown of DUB3 greatly inhibited the 
migratory ability and invasiveness of MDA-MB231 cells and suppressed lung 
colonization using an experiment MDA-MB-231 metastases model. Liu et al. went 
on to show that CDK4/6 phosphorylates DUB3 at Ser41, thus activating DUB3. 
Treatment of cells with palbociclib inhibited DUB3 activity, decreased SNAIL and 
vimentin expression and decreased cell migration. These findings were confirmed 
with the drug abemaciclib. In a highly metastatic TNBC patient-derived xenograft 
(PDX) model derived from the Mayo BEAUTY study, the administration of 
palbociclib did not alter primary tumor growth but significantly reduced lung and 
liver metastases. The findings of Liu et al were additionally validated in a 
companion paper published at the same time by Wu et al.33 Additionally, in a 
separate laboratory, Qin et al demonstrated that the EMT markers, vimentin and 
Snail, were down-regulated with palbociclib treatment in the MDA-MB231 
model.34 
While current SNAIL antibodies are not robust for clinical application, taken 
together, these data identify a potential new CDK4/6i drug target and suggest that 
changes in expression of EMT targets such as SNAIL and vimentin be predictive of 
CDK4/6i drug effect. 
 Effects on Tumor Grade 
Regarding the phenotype of 
tumor grade, Martin et al 
recently presented the results 
of the NeoMonarch clinical 
study evaluating neoadjuvant 
abemaciclib alone or in 
combination with aromatase 
inhibitors for early stage 
ER+/HER2- breast cancer.33 In 
a blinded analysis, abemaciclib 
monotherapy for 2 weeks 
reduced tumor grade in 13/46 
patients (28%) compared to 
anastrozole alone, which 
reduced tumor grade in 5/49 
patients (10%). Similar 
observations with regard to a 
change in tumor histological phenotype have been observed in vitro and in vivo. 
Specifically Goetz et al demonstrated that short term abemaciclib (5 days) altered 
organoid formation in MDA 436 cells (TNBC, claudin low) while Goel et al 
demonstrated that prolonged exposure to abemaciclib in vivo (MMTV ER+/HER2+ 
transgenic tumor) resulted in a more differentiated phenotype (Figure 1). 
 
Figure 1. Unpublished data regarding the effects of abemaciclib on 
5-day organoid formation (MDA 436)(Goetz) and after prolonged 
in vivo abemaciclib treatment (MMTV-rtTA/tetO-HER2 transgenic 
tumors) (Goel)  

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 Effects of CDK4/6 inhibitors in LAR expressing TNBC 
Finally, Asghar et al. demonstrated that the luminal androgen receptor (LAR) 
subtype of TNBC was highly sensitive to CDK4/6 inhibition,35 potentially 
extending the use of CDK 4/6i to the LAR subtype, a TNBC subset that is 
considered resistant to chemotherapy. 
3.4 Clinical importance of immune and EMT biomarkers in TNBC. 
Miyashita et al demonstrated that following completion of NAC for TNBC, >50% of 
tumors had a CD8/FOXP3 ratio of 1.6 or higher or a fold change of 1.8 in the 
CD8/FOXP3 ratio (that is, ratio of post-NAC CD8/FOXP3 ratio value to pre-NAC 
CD8/FOXP3 ratio).36 In these patients, recurrence-free survival (RFS) and breast-
cancer specific survival (BCSS) were significantly improved for those with post-NAC 
CD8/FOXP3 ratio of 1.6 or higher or a fold change of 1.8 or higher in the CD8/FOXP3 
ratio. Based on preliminary data from the laboratory of Goel et al demonstrating 
abemaciclib induced suppression of FOXP3 Tregs, our hypothesis is that abemaciclib 
will suppress Tregs and thus increase the CD8/FOXP3 ratio, a finding associated with 
improved BCSS in TNBC. 
Karihtala et al reported that 65% of women with TNBC have vimentin expressing 
invasive cancer cells (negative: 0-1%; 1+: 2-9%; 2+: 10-50%; 3+: over 50%) and that 
vimentin over-expression (2+/3+) is associated with poor breast cancer specific 
survival.37 However, there are no data regarding the frequency of vimentin expression 
in TNBC with residual disease after standard NAC. Therefore, using a validated IHC 
antibody (Dako (M7020, clone 3B4) and as assessed in the Mayo CAP/CLIA 
laboratory, we identified a cohort of 30 Mayo clinic patients who underwent NAC with 
AC and taxane for TNBC (ER <1%, PR <1% and HER2 negative) and who had residual 
disease. Of these, 22 who FFPE sections available from residual disease at surgery, and 
15/22 (68%) had strong vimentin staining (3+) in the residual tumor specimens. These 
data support the observation that EMT markers such as vimentin are highly expressed 
in TNBC resistant to chemotherapy. 
In summary, diverse and newly described effects of CDK4/6i provide the rationale to 
prospectively study the pharmacodynamics of abemaciclib in TNBC using a “window” 
design in a chemotherapy resistant population of breast cancer patients treated either 
with or without pembrolizumab. Specifically, for TNBC patients who have completed 
NAC +/- pembrolizumab and have biopsy proven residual disease, we propose a 
window trial, to prospectively study the pharmacodynamics effects of abemaciclib on 
1) CD8 T cells and FOXP3 regulatory cells (increase CD8/FOX3 ratio); 2) EMT 
pathway markers including vimentin, SNAIL/SLUG, TWIST, and pDUB3; 3) 
proliferation biomarkers (Ki-67); and 4) the expression of interferon-stimulated genes 
and genes involved in antigen presentation. Each of these parameters will be evaluated 
within the known TNBC molecular subtypes and based on known Rb status. The effects 
of abemaciclib will be evaluated in two different cohorts of patients treated either 
without pembrolizumab (Cohort A) or with pembrolizumab (Cohort B). 
If our trial is successful, and abemaciclib is found to activate immune targeting T-Cells 
or inhibit metastases though direct effects on EMT in chemotherapy resistant TNBC, 

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there would be clear rationale for development of abemaciclib in the adjuvant treatment 
of TNBC. 
3.5 Mayo Clinic Prospective Neoadjuvant Chemotherapy Study 
Mayo investigators funded by the Center for Individualized Medicine and the Cancer 
Center developed a prospective clinical trial which incorporated treatment of high 
risk breast cancers with standard neoadjuvant chemotherapy along with collection of 
serial tumor samples for sequencing and development of PDX to further characterize 
tumor factors impacting response to chemotherapy.3,38 The BEAUTY study enrolled 
140 patients from March 2012 through May 2014 across all three (3) Mayo sites. 
Baseline tumor biopsies were obtained followed by treatment with a combination of 
taxane (with anti-HER2 therapy if HER2+ disease) and 
anthracycline/cyclophosphamide chemotherapy. Thirty-four percent of patients 
enrolled had TNBC. While 53% of the TNBC patients demonstrated pCR, 47% of 
patients had some degree of residual disease and therefore are at high risk of relapse. 
3.6 Abemaciclib (Verzenio®) 
There are currently 3 CDK 4/6 inhibitors that have demonstrated evidence for benefit 
in metastatic ER+, HER2- breast cancer. These include palbociclib, ribociclib, and 
abemaciclib, all of which are FDA approved for ER+ metastatic breast cancer. 
 Preclinical  
Abemaciclib is a CDK4 and 6 inhibitor with three FDA indications for the treatment 
of hormone receptor-positive (HR+), human epidermal growth factor receptor 2 
negative (HER2−) advanced or metastatic breast cancer (ABC): In combination with 
letrozole or anastrozole for postmenopausal women as initial endocrine−based 
therapy (MONARCH 3), In combination with fulvestrant for women with disease 
progression following endocrine therapy (MONARCH 2), and as a single agent for 
adult patients with disease progression following endocrine therapy and prior 
chemotherapy in the metastatic setting (MONARCH 1). This compound differs from 
both ribociclib and palbociclib in terms of its chemical structure and pharmacological 
characteristics.39 It can be administered continuously and is able to cross the blood-
brain barrier. 
 Clinical 
Initial data regarding the clinical pharmacology, safety, and efficacy of abemaciclib 
in humans came from the JPBA study, an open-label, phase 1, dose escalation 
study, followed by tumor-specific expansion cohorts.40 During the dose escalation 
phase, abemaciclib was administered at doses of 50–225 mg daily and 75– 275 mg 
twice daily. The recommended dose for phase II evaluation (RP2D) was 200 mg 
twice daily and a dose-proportional pharmacokinetic profile was noted, with a half-
life ranging from 17 to 38 hours. Pharmacodynamic assessments were conducted in 
tumors and skin keratinocytes. Despite the long half-life, partial reversibility of 
abemaciclib-induced Rb phosphorylation and topoisomerase II-α expression in skin 
biopsies taken from patients treated once daily was observed. As the twice-daily 

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schedule demonstrated evidence of sustained target engagement over the dosing 
interval, this was further evaluated in the tumor-specific cohorts. 
Fatigue was the dose-limiting toxicity of abemaciclib, which differed from that of 
palbociclib and ribociclib (neutropenia). Frequently observed treatment-related 
adverse events in the single-agent tumor-specific arms (n = 173) included fatigue 
(all grades = 41%; Grade 3 = 3%), diarrhea (all grades = 63%; Grade 3 = 5%), 
nausea (all grades = 45%; Grade 3 = 2%), vomiting (all grades = 25%; Grade 3 = 
1%), and anorexia (all grades = 17%; Grade 3 = 0 %). Although diarrhea was a 
common side effect, it was effectively managed with prophylactic medications or a 
dose reduction. Hematologic side effects were less commonly observed when 
compared with palbociclib and ribociclib, e.g. leukopenia (all grades = 25%; Grade 
3 = 10%), neutropenia (all grades = 23%; Grade 3 = 9%; Grade 4 = 1%), 
thrombocytopenia (all grades = 23%; Grade 3 = 7%), and anemia (all grades = 
20%; Grade 3 = 4%). JPBA also included a monotherapy expansion cohort (n=47) 
of heavily pretreated metastatic breast cancer (MBC) patients (median of 7 prior 
lines of systemic therapy). Given the low-grade diarrhea that complicated the 
200 mg twice-daily dose level, some patients received a lower starting dose of 
150 mg twice daily which was associated with fewer treatment delays and dose 
reductions. Robust antitumor activity was observed including patients with HR-
positive/HER2 negative breast cancer as well as HR+/HER2-positive MBC.  
Preclinical data have demonstrated that abemaciclib effectively penetrates the BBB 
and improves survival outcomes in intracranial human brain tumor xenografts.41 
Therefore, paired cerebrospinal fluid (CSF) and unbound plasma concentrations of 
abemaciclib were procured from ten patients with glioblastoma participating in the 
JPBA trial. It was demonstrated that the CSF concentration of abemaciclib was 
similar to the plasma concentration of abemaciclib. Further, two patients with 
glioblastoma treated with abemaciclib achieved a prolonged PFS, which suggested 
that abemaciclib may be effective in the treatment of primary or metastatic brain 
tumors, and clinical trials are in progress. A phase II trial is studying the activity of 
abemaciclib in patients with brain metastases, including in a cohort of patients with 
HR+/HER2- advanced breast cancer.42 Of 23 evaluable patients with HR+/HER2- 
MBC, two patients had confirmed durable partial responses in their brain lesions, 
and more patients are now being enrolled in the second phase of the study to further 
determine the potential benefit of CDK4/6i in patients with brain metastases. Of 
note, no responses were observed in patients with brain metastases secondary to 
HR+, HER2+ breast cancer. 
The safety and efficacy of abemaciclib monotherapy was further evaluated in the 
phase 2 single-arm MONARCH 1 study (NCT02102490).5 Women (n=132) with 
locally advanced or metastatic HR-positive, HER2-negative breast cancer whose 
disease had progressed following both endocrine therapy and 1-2 lines of 
chemotherapy in the metastatic setting were enrolled and treated with abemaciclib 
200 mg every 12 hours until disease progression. The ORR was 19.7%, the clinical 
benefit rate (CR + PR + SD 6 months) was 42.4%, and median PFS was 
6.0 months. The most frequently observed treatment-related adverse events were: 
creatinine elevation (all grades = 98.5%; grade 3 = 0.8%), diarrhea (all grades = 

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90.2%; Grade 3 = 19.7%), neutropenia (all grades = 87.7%; Grade 3/4 26.9%) and 
fatigue (all grades = 65.2%; Grade 3 = 12.9%). As abemaciclib is a competitive 
inhibitor of creatinine efflux transporters (OCT2, MATE1, and MATE2-K), 
cystatin C calculated glomerular fraction rate was performed and values were 
within normal limits. Notably, abemaciclib-induced diarrhea was manageable with 
antidiarrheal medication or dose reduction, and discontinuation of therapy due to 
side-effects was infrequent (7.6%). Notably, the results of this trial resulted in FDA 
approval. 
Several trials have evaluated the combination of abemaciclib and endocrine 
therapy, including the JPBA trial (abemaciclib 200 mg twice daily combined with 
fulvestrant40 and the phase 1b JPBH study (NCT02057133). In the latter, the safety, 
pharmacokinetics, and antitumor activity of abemaciclib in combination with 
various endocrine therapies and HER2-directed therapy has been evaluated. 
Participants were allocated to one of six cohorts and received either abemaciclib 
plus letrozole 2.5 mg daily (part A), anastrozole 25 mg daily (part B), tamoxifen 
20 mg daily (part C), exemestane 25 mg daily (part D), exemestane 25 mg daily 
plus everolimus 5 mg daily (part E), or trastuzumab 6–8 mg/kg IV once every 
21 days (part F). Participants in parts A–E had HR-positive, HER2-negative MBC 
and those in part F had HER2-positive MBC. Patients in parts A-E could not have 
received prior chemotherapy in the metastatic setting, however one prior line of 
chemotherapy was allowed for metastatic disease in part F. Preliminary data from 
the first 65 patients included in parts A–D have been presented and demonstrated 
that the most common treatment- related adverse events were diarrhea (all grades = 
95%; Grade 3 = 31%), fatigue (all grades = 71%; Grade 3 = 14%), nausea (all 
grades = 62%; Grade 3 = 6%), and neutropenia (all grades = 31%; Grade 3 = 17%). 
Diarrhea was manageable with antidiarrheal agents or dose reduction. The disease 
control rate (CR + PR + SD) was 67% for parts A + B (non-steroidal aromatase 
inhibitors (36 patients), including 2 confirmed PRs, and 75% for part C (tamoxifen 
16 patients). Updated results from this study were recently reported. In part F, 
eleven patients had HR-positive HER2-positive MBC and seven patients had HR-
negative, HER2-positive MBC. Interestingly, although no complete responses (CR) 
or partial responses (PR) were noted in the patients with HR-negative, HER2-
positive disease, four patients achieved stable disease (SD). Further, in the 
7 patients who were treated with trastuzumab and a 150mg dose of abemaciclib, the 
disease control rate (CR + PR + SD) was comparable in patients with HR-positive 
and HR-negative disease (54.5 % vs. 57.1 %, respectively). These data suggest that 
further study of abemaciclib in ER-negative breast cancer is warranted.  
In the FDA registration phase III double-blind MONARCH 2 study , 669 patients 
with HR-positive, HER2-negative advanced breast cancer who had progressed 
while receiving neoadjuvant/adjuvant endocrine therapy ET, ≤12 months from the 
end of adjuvant ET, or while receiving first-line ET for metastatic disease, were 
enrolled.10 Patients were randomized in a 2:1 fashion to receive fulvestrant 
(standard dosing) along with either abemaciclib (150 mg twice daily) or placebo. 
The combination of abemaciclib and fulvestrant significantly extended PFS versus 
fulvestrant alone (median, 16.4 v 9.3 months; hazard ratio, 0.553; 95% CI, 0.449 to 

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0.681; P <0.001) with higher response rates observed in patients with measurable 
disease (48.1% (95% CI, 42.6% to 53.6%) vs. 21.3% (95% CI, 15.1% to 27.6%). 
The most frequent adverse events in the abemaciclib versus placebo arms were 
diarrhea (86.4% vs 24.7%), neutropenia (46.0% vs 4.0%), nausea (45.1% vs 
22.9%), and fatigue (39.9% vs 26.9%). In 2019, a formal survival analysis was 
performed. Patients randomized to abemaciclib plus fulvestrant had superior 
overall survival compared to fulvestrant alone (HR 0.757; p=0.0137; 46.7 months 
vs 37.3 months). Most notably, in patients with primary endocrine resistance, 
abemaciclib extended survival compared to the placebo arm by 7.2 months (HR 
0.686; 38.7 months vs 31.5 months).56 
The double-blind phase III MONARCH 3 FDA Registration trial randomized 493 
patients to 150 mg of abemaciclib or placebo twice daily combined with either 1 mg 
of anastrozole or 2.5 mg of letrozole daily until disease progression or unacceptable 
toxicity.9 The study included postmenopausal women with locoregionally recurrent 
or metastatic breast cancer and no prior systemic treatment for advanced disease. 
Median PFS was increased in those who received abemaciclib (P = .000021; 
median: not reached in the abemaciclib arm, 14.7 months in the placebo arm). For 
participants with measurable disease, the objective response rate (ORR) was 59% in 
the abemaciclib group and 44% in the placebo arm (P = .004). Diarrhea was the 
most frequent adverse effect (81.3%) observed in patients who received 
abemaciclib, but was usually Grade 1 (44.6%). With regards to differences in 
toxicity between patients who received abemaciclib vs. placebo, the most frequent 
Grade 3 or 4 adverse events were neutropenia (21.1% v 1.2%), diarrhea (9.5% v 
1.2%), and leukopenia (7.6% v 0.6%). The most common AEs overall (of any 
grade) were diarrhea, neutropenia, fatigue, and nausea. Notably, there was an 
increased incidence of venous thromboembolic disease in the abemaciclib arm (in 
16 (4.9%) of patients versus one (0.6%) in the placebo arm).9,10,43 Safety of CDK4/6 
inhibitors in the pre-operative window setting. 
A recent palbociclib window study demonstrated that 2 weeks of treatment was 
safe with no delay in surgery, and palbociclib significantly decreased Ki-67.44 
3.7 Assessing response to neoadjuvant chemotherapy 
Neoadjuvant chemotherapy decreases tumor burden in the breast and the axillary 
lymph nodes. Assessment of the extent of residual disease after neoadjuvant 
chemotherapy is critical to evaluate response to chemotherapy and to guide surgical 
decision making. Pathologic complete response (pCR) defined as no residual 
invasive disease in the breast or the lymph nodes occurs in approximately 30-40% 
of all cases45with higher rates of approximately 40-50% in patients with TNBC.46  
Assessment of extent of residual disease in the breast can be performed using 
mammogram, ultrasound and/or MRI. Ultrasonography is more accurate than 
clinical breast examination or mammography for assessing residual tumor size 
following NAC.47 When both mammogram and ultrasound demonstrate no residual 
disease the likelihood of a pCR was 80 %.48 Breast MRI has the greatest accuracy 
in assessing extent of residual disease. However MRI also may overestimate and 
underestimate the presence and extent of residual disease. At the current time, 

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given the limitations of breast imaging to reliably demonstrate the presence or 
absence of residual disease after NAC, histological evaluation of the tumor bed is 
standard of care. In this study, a percutaneous biopsy will be performed to obtain 
tumor tissue to prove there is residual invasive disease and identify patients that 
would candidates for the neoadjuvant window study. In order to increase the 
likelihood of identifying the area of the tumor biopsied at pre-registration, a clip 
will be placed at the time of the biopsy. Identification of the clip and collection of 
tumor tissue around the clip will be mandated to increase the likelihood of 
collection of tumor in the same region (pre/post abemaciclib). 
3.8 Trial Summary 
Nearly all patients with TNBC are being treated with neoadjuvant chemotherapy 
either with or without pembrolizumab [ref: PMID: 32101663]. However, multiple 
studies have demonstrated that TNBC patients with residual disease after receipt of 
standard neoadjuvant based chemotherapy have high rates of distant recurrence and 
death within 3 years. Therefore, prioritization of drugs that target this group of 
patients is of high priority. 
The neoadjuvant window trial design has been successfully utilized to develop 
multiple drugs such as aromatase inhibitors. However, the use of a window design 
to test drugs in patients with chemotherapy resistant disease has not been explored. 
A major issue for developing drugs in the post-neoadjuvant space relates to the lack 
of tissue for examining drug response phenotypes. The development of a “window” 
trial in the post-neoadjuvant space allows the interrogation of tumor tissue that is 
resistant to chemotherapy and provides the opportunity to study biomarkers 
associated with critical drug response phenotypes. Therefore, the use of a post-
neoadjuvant window is novel, and provides an important opportunity to develop a 
new approach to drug development. 
Abemaciclib is FDA approved for the treatment of estrogen receptor positive, 
HER2 negative metastatic breast cancer. However, multiple publications suggest 
that drugs within this class may have antitumor effects through a broad range of 
mechanisms, including effects on immune activation, effects on EMT, and 
proliferation (e.g. for LAR breast cancer with intact Rb). Therefore, our proposed 
study designed to assess abemaciclib response phenotypes in TNBC resistant to 
chemotherapy and/or immune checkpoint therapy, could provide critical 
information for development of larger studies to evaluate the antitumor activity of 
abemaciclib in TNBC. For example, if abemaciclib is found to activate immune 
signatures, alter tumor signatures associated with distant metastases, or inhibit 
proliferation, abemaciclib may demonstrate to be an important adjuvant treatment 
to improve the survival of women with chemotherapy resistant TNBC. 
3.9 Lowering of Starting Dose of Abemaciclib 
The dosing and adverse event data of the first 4 patients treated with abemaciclib 
were reviewed in early May 2021. Two of these 4 patients discontinued treatment 
due to adverse events after 12 and 14 days of treatment. The first patient 
discontinued treatment (5 days prior to surgery) having developed Grade 2 diarrhea 

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and vomiting with Grade 1 nausea, fatigue, and anorexia. The second patient 
discontinued treatment (8 days prior to surgery) having developed Grade 1 diarrhea, 
nausea, alanine aminotransferase increase, and platelet count decrease. Given that 
the goal of this window study is to investigate the pharmacodynamic effects of 
abemaciclib in surgically resected tumor tissue, adherence to abemaciclib dosing up 
to the day prior to surgery is critical to meet the trial endpoints. Thus, given these 
tolerability issues, the protocol was amended to lower the starting dose of 
abemaciclib from 200mg every 12 hours to 150 mg every 12 hours. 
3.10 Addition of Cohort B (evaluating effects of abemaciclib in TNBC patients 
treated with pembrolizumab and NAC) 
Based on the preclinical data outlined above, phase I studies were conducted to 
evaluate the toxicities and antitumor activity of the combination of abemaciclib 
and pembrolizumab [DOI: 10.1158/1538-7445.AM2020-CT108]. However, 
further clinical development was halted because of high rates of Grade 3/4 
toxicity when the drugs were combined. In spite of this issue, there remains strong 
rationale to study abemaciclib sequentially in TNBC patients who exhibit residual 
disease after receipt of combination NAC and pembrolizumab. Therefore, we will 
add a second cohort of TNBC patients who were treated with NAC plus 
pembrolizumab, based on the recent FDA approval of the use of pembrolizumab 
along with NAC as previously published in the Keynote 522 study 
[https://www.nejm.org/doi/full/10.1056/NEJMoa1910549]. 
 
 
 

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4. PARTICIPANT SELECTION 
4.1 Pre-Registration Inclusion Criteria 
 Women of age ≥18 years. (NOTE: Rationale provided in Section 17.8.) 
 Clinical T1-4, N0-3, M0 breast cancer at diagnosis (prior to the start of 
neoadjuvant chemotherapy) by AJCC staging version 8. 
Note: Benign breast disease, lobular carcinoma in situ (LCIS) or ductal 
carcinoma in situ (DCIS) in the ipsilateral or contralateral breast is 
allowed. 
Note: Additional ipsilateral or contralateral invasive breast cancer is 
allowed. The index lesion is the largest triple-negative, chemotherapy-
resistant lesion. 
 Histological confirmation of triple negative invasive breast cancer (defined 
as ER≤10%, PR≤10% and HER2 not amplified by ISH or IHC 0/1) at 
diagnosis. 
 Neoadjuvant chemotherapy (NAC) 
 Cohort A: CLOSED TO PRE-REGISTRATION and 
REGISTRATION as of MCCC Amendment 6 
Neoadjuvant chemotherapy (NAC) with one of the following 
regimens that was not discontinued early due to intolerability with less 
than 50% of planned treatment given due to disease progression or 
patient request:  
- Paclitaxel or docetaxel followed by one of the following: the 
combination of doxorubicin and cyclophosphamide (AC); the 
combination of epirubicin and cyclophosphamide (EC) or the 
combination of 5-fluorouracil, epirubicin and cyclophosphamide 
(FEC) 
Note: Carboplatin may be added to these regimens 
- AC or EC or FEC followed by docetaxel or paclitaxel 
Note: Carboplatin may be added to these regimens 
- Docetaxel in combination with doxorubicin and cyclophosphamide 
(TAC)  
- Docetaxel in combination with cyclophosphamide (TC) (for patients 
who are not candidates for anthracyclines) 
- Carboplatin or cisplatin in combination with a taxane (paclitaxel, 
docetaxel, or nab-paclitaxel) (for patients who are not candidates for 
anthracyclines) 
 Cohort B: Neoadjuvant chemotherapy (NAC) with one of the 
following regimens in combination with pembrolizumab that was not 
discontinued early due to intolerability with less than 50% of planned 

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treatment given due to disease progression or patient request:  
- Paclitaxel or docetaxel followed by one of the following: the 
combination of doxorubicin and cyclophosphamide (AC); the 
combination of epirubicin and cyclophosphamide (EC) or the 
combination of 5-fluorouracil, epirubicin and cyclophosphamide 
(FEC) 
Note: Carboplatin may be added to these regimens 
- AC or EC or FEC followed by docetaxel or paclitaxel [Note: 
Carboplatin may be added to these regimens] 
- Docetaxel in combination with doxorubicin and cyclophosphamide 
(TAC) 
- Docetaxel in combination with cyclophosphamide (TC) 
- Carboplatin or cisplatin in combination with a taxane (paclitaxel, 
docetaxel, or nab-paclitaxel) 
 Residual lesion/enhancement seen in the breast on breast imaging performed 
after completion of NAC. 
 Able to swallow oral medication. 
 Willing to undergo biopsy for research (Sections 5 and 10). 
 Willing to provide tissue and blood samples for correlative research purposes 
(see Section 10).  
 Willing to stop use of strong and moderate inducers and/or strong inhibitors 
of cytochrome P450 3A ≤7 days prior to registration. 
 Provide written informed consent. 
4.2 Pre-Registration Exclusion Criteria 
 History of deep venous thrombosis (DVT) or pulmonary embolisms (PE) ≤12 
months prior to preregistration; OR 
Active DVT and/or PE requiring anti-coagulant therapy. 
NOTE: Patients who are on anti-coagulant therapy for maintenance are 
eligible as long as the DVT and/or PE was >12 months prior to enrollment 
and there is no evidence for active thrombosis (either DVT or PE).  
NOTE: Patients on anticoagulation are eligible; however peri-biopsy and 

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peri-surgical management of anticoagulation is per the institutional standard 
of care. 
 Prior treatment with CDK 4/6 inhibitors (e.g. palbociclib, ribociclib, 
abemaciclib, etc.) 
 Prior treatment with radiation for this breast cancer.  
 Prior incisional or excisional breast biopsy for this cancer. 
 Any contraindications to pre-registration biopsy (such as bleeding diatheses, 
etc.). 
 Receiving any investigational agent which would be considered as a 
treatment for the primary neoplasm. 
 Other active malignancy ≤3 years prior to registration.  
EXCEPTIONS: Non-melanotic skin cancer or carcinoma-in-situ of the 
cervix.  
NOTE: If there is a history of prior malignancy, they must not be receiving 
another specific treatment for prior malignancy.  
 Biopsy proven Stage IV breast cancer. 
 Serious pre-existing medical conditions that would preclude participation in 
this study (for example, interstitial lung disease, severe dyspnea at rest or 
requiring oxygen therapy, severe renal impairment [e.g., estimated creatinine 
clearance <30ml/min], history of major surgical resection involving the 
stomach or small bowel, or preexisting Crohn’s disease or ulcerative colitis 
or a preexisting chronic condition resulting in baseline Grade 2 or higher 
diarrhea). 
 History of any of the following conditions:  
• Syncope of cardiovascular etiology 
• Ventricular arrhythmia of pathological origin (including, but not 
limited to, ventricular tachycardia and ventricular fibrillation) 
• Sudden cardiac arrest 
NOTE: Patients on anticoagulation are eligible; however peri-biopsy and 
peri-surgical management of anticoagulation is per the institutional 
standard of care. 
4.3 Registration - Inclusion Criteria for All Patients 
 Registration must occur ≤56 days after last dose of NAC. 
 ECOG Performance Status (PS) 0, 1, or 2 (Appendix A) 
 Cohort B Group 4 ONLY:  
 The following laboratory values obtained after completion of NAC but 
≤14 days prior to registration: 
• Absolute neutrophil count (ANC) ≥1500/mm3 

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• Platelets (PLT) ≥100,000/mm3 
• Hemoglobin (HgB) ≥8.0g/dL 
• Total bilirubin ≤1.5 x ULN 
• Aspartate transaminase (AST)(SGOT) ≤3 x ULN 
• Alanine aminotransferase (ALT)(SGPT) ≤3 x ULN 
• Serum creatinine ≤1.5 x ULN 
 Negative pregnancy test done ≤7 days prior to registration, for persons of 
childbearing potential only. 
4.4 Registration - Exclusion Criteria for Cohort B Group 4 only 
 Any of the following because this study involves an investigational agent 
whose genotoxic, mutagenic and teratogenic effects on the developing fetus 
and newborn are unknown: 
• Pregnant persons 
• Nursing persons 
• Persons of childbearing potential who are unwilling to employ 
adequate contraception 
 Failure to recover to Grade 1 or lower from effects of neoadjuvant 
chemotherapy. 
Exceptions: Residual alopecia and Grade 2 peripheral neuropathy are 
allowed. 
 Concurrent use of strong and moderate inducers and/or strong inhibitors of 
cytochrome P450 3A ≤7 days prior to registration. 
 Known infections as follows (NOTE: Screening is not required for 
enrollment): 
• Active systemic bacterial infection requiring intravenous antibiotics 
• Active fungal infection (requiring intravenous or oral antifungal 
treatment) 
• Detectable viral infections (e.g., known HIV, known active hepatitis B 
or C) 
 Concurrent use of chemotherapy, radiotherapy, immunotherapy, or other 
components of neoadjuvant treatment. 
NOTE: Patients must complete all elements of NAC ≥21 days prior to 
starting abemaciclib. 
4.5 Inclusion of Underrepresented Populations 
Individuals of all races and ethnic groups are eligible for this trial. There is no bias 
towards age or race in the clinical trial outlined. This trial is open to the accrual of 
women only. 

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5. REGISTRATION PROCEDURES 
5.1 Grouping Factors for Eligiblity Checklist 
 Disease group: No invasive disease (Group 3) vs. Invasive disease (Group 4) 
NOTE: Groups 1 and 2 are closed as of Amendment 6 
 Cohort: Cohort A (neo-adjuvant treatment did not include pembrolizumab) 
vs. Cohort B (neo-adjuvant treatment included pembrolizumab) 
NOTE: Cohort A is closed as of Amendment 6 
5.2 Pre-Registration (Step 0)  
To pre-register a patient, access the Mayo Clinic Research Registration Application at 
https:\\registration.mayo.edu. The Research Registration Application is available 24 
hours a day, 7 days a week. Back up and/or system support contact information is 
available on the website. If unable to access the website, call the Research 
Registration Office at (507) 284-2753 or random01@mayo.edu between the hours of 
8 a.m. and 5:00 p.m. Central Time (Monday through Friday). 
The instructions for the Research Registration Application are available on the Office 
of Clinical Trials web page ((https://www.mayo.edu/research/centers-
programs/center-clinical-translational-science/offices/office-of-clinical-
trials/research-registration-application) and detail the process for completing and 
confirming patient registration. Prior to initiation of protocol treatment, this process 
must be completed in its entirety and a subject ID number must be available as noted 
in the instructions. It is the responsibility of the individual and institution registering 
the patient to confirm the process has been successfully completed prior to release of 
the study agent. Patient registration via the Research Registration Application can be 
confirmed in any of the following ways: 
• Contact the Research Registration Office (507) 284-2753 or 
random01@mayo.edu. If the patient was fully registered, the Research 
Registration Office staff can access the information from the centralized database 
and confirm the registration. 
• Refer to “Instructions for Remote Registration” in section “Finding/Displaying 
Information about A Registered Subject.” 
 Verification at pre-registration 
Prior to accepting the pre-registration, the registration/randomization application 
will verify the following: 
• IRB approval at the registering institution 
• Patient pre-registration eligibility  
• Existence of a signed consent form  
• Existence of a signed authorization for use and disclosure of protected health 
information. 
 Pre-registration tests/procedures  
Pre-registration tests and procedures (see Section 6.0) must be completed within 
the guidelines specified on the test schedule. 

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5.3 Registration Procedures 
 Registration 
To register a patient, access the Mayo Clinic Research Registration Application at 
https:\\registration.mayo.edu. The Research Registration Application is available 24 
hours a day, 7 days a week. Back up and/or system support contact information is 
available on the website. If unable to access the website, call the Research 
Registration Office at (507) 284-2753 or random01@mayo.edu between the hours of 
8 a.m. and 4:30 p.m. Central Time (Monday through Friday). 
The instructions for the Research Registration Application are available on the Office 
of Clinical Trials web page ((https://www.mayo.edu/research/centers-
programs/center-clinical-translational-science/offices/office-of-clinical-
trials/research-registration-application) and detail the process for completing and 
confirming patient registration. Prior to initiation of protocol treatment, this process 
must be completed in its entirety and a subject ID number must be available as noted 
in the instructions. It is the responsibility of the individual and institution registering 
the patient to confirm the process has been successfully completed prior to release of 
the study agent. Patient registration via the Research Registration Application can be 
confirmed in any of the following ways: 
• Contact the Research Registration Office (507) 284-2753 or 
random01@mayo.edu. If the patient was fully registered, the Research 
Registration Office staff can access the information from the centralized database 
and confirm the registration. 
• Refer to “Instructions for Remote Registration” in section “Finding/Displaying 
Information about A Registered Subject.” 
 Correlative Research  
A mandatory tissue and blood correlative research component is part of this 
study. The patient will be automatically registered onto this component (see 
Section 10). 
 Documentation 
Documentation of IRB approval must be on file in the Research Registration 
Office before an investigator may register any patients. 
In addition to submitting initial IRB approval documents, ongoing IRB approval 
documentation must be on file (no less than annually) at the Research Registration 
Office (fax: 507-284-0885 or email: random01@mayo.edu). If the necessary 
documentation is not submitted in advance of attempting patient registration, the 
registration will not be accepted and the patient may not be enrolled in the 
protocol until the situation is resolved. 
When the study has been permanently closed to patient enrollment, submission of 
annual IRB approvals to the Research Registration Office is no longer necessary. 
 Verification at registration 
Prior to accepting the registration, registration/randomization application will 
verify the following: 

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• IRB approval at the registering institution 
• Patient eligibility 
• Existence of a signed consent form 
• Existence of a signed authorization for use and disclosure of protected health 
information  
 Patient permissions 
At the time of registration, the following will be recorded: 
• Patient has/has not given permission to store and use his/her sample(s) for 
future research on cancer. 
• Patient has/has not given permission to store and use his/her sample(s) for 
future research to learn about, prevent, or treat other health problems. 
• Patient has/has not given permission for someone to contact them about future 
participation in more research 
 Additional Qualifications 
 Treatment on this protocol must commence at Mayo Clinic under the 
supervision of a medical oncologist. 
 Treatment cannot begin prior to registration and must begin ≤14 days 
after registration. 
 Pretreatment tests/procedures (see Section 6) must be completed within 
the guidelines specified on the test schedule. Prior to registration a 
research biopsy of breast tissue must be taken for central pathologic 
review. 
 All required baseline symptoms must be documented and graded. 
 Cohort B Group 4 ONLY: Study drug is available on site. 
 Blood draw kit is available on site. 
 Stool kit is available on site. 
 

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6. STUDY CALENDAR 
6.1 Cohort B GROUP 3 Study Calendar – No invasive disease 
 
 
 
 
Parameter 
Pre-Reg 
Registration
1 
(Baseline) 
At time of 
surgery 
Demographics 
 
X 
 
CLINICAL EVALUATIONS: 
 
 
 
History and Physical including vital signs2, weight, PS 
 
X 
 
Height 
 
X 
 
LABORATORY/RADIOLOGIC EVALUATIONS: 
 
 
 
Hematology (CBC with diff) 
X3 
  
 
Tumor measurement (breast imaging) and report4 
X 
 
 
CORRELATIVE STUDIES: 
 
 
 
Research Tumor/Tissue Biopsy 
X 
 
 
Research tissue specimens from clinical procedures 
X5 
 
X 
Research blood specimens 
 
X6 
 
Research stool specimens 
 
X7 
 
ADDITIONAL INFORMATION: 
 
 
 
Submission of Surgical (Operative) and Pathology Reports 
X8 
 
X 
 
1 ≤14 days prior to registration 
2 Vital signs include blood pressure, pulse, temperature 
3 After completion of NAC and ≤3 days prior to biopsy to rule out Grade 4 neutropenia 
4 Tumor measurement/breast imaging should be per institutional standard of care. Bi-dimensional measurement 
should be collected. Imaging reports from diagnosis and post-NAC should be submitted for all patients during 
pre-registration. 
5 Collection of a representative block or slides from initial diagnostic biopsy (prior to neoadjuvant chemotherapy) 
6 Research blood draw must be scheduled (paid by study); May be drawn after patient consents to the study and pre-
registration patient study ID is available 
7 Baseline stool collection kit may be given to patient any time after consent is obtained and pre-registration patient 
study ID is available. 
8 Submit reports from biopsy at the time of diagnosis as well as reports from Pre-Registration research biopsy for 
this study 

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6.2 Cohort B GROUP 4 Study Calendar – Residual invasive disease 
 
 
 
 
 
 
 
Parameter 
Pre-Reg 
Registration9 
(Baseline) 
Abemaciclib 
14-21 days 
prior to surgery 
Prior to 
surgery 
At time of 
surgery 
End of Study 
30-60 days 
after surgery10 
Demographics 
 
X 
 
 
 
 
CLINICAL EVALUATIONS: 
 
 
 
 
 
 
History and Physical including vital signs11, weight, PS 
 
X 
 
X 
 
 
Height 
 
X 
 
 
 
 
LABORATORY/RADIOLOGIC EVALUATIONS: 
 
 
 
 
 
 
Hematology (CBC with diff) 
X12 
X 
 
X13 
 
 
Chemistries14 
 
X 
 
X 
 
 
Pregnancy Test (serum or urine)15 
 
X 
 
 
 
 
Tumor measurement (breast imaging) and report16 
X 
 
 
 
 
 
TREATMENT ADMINISTRATION: 
 
 
 
 
 
 
Abemaciclib 
 
 
X 
 
 
 
 
9 ≤14 days prior to registration 
10 Telephone contact approximately 30-60 days after definitive surgery (breast tumor resection) 
11 Vital signs include blood pressure, pulse, temperature 
12 After completion of NAC and ≤3 days prior to biopsy to rule out Grade 4 neutropenia 
13 CBC with differential ≤3 days prior to surgery (This panel and venipuncture are covered by research.) 
14 Serum chemistry includes a complete metabolic panel (CMP) [CPT 80053]  
15 For persons of childbearing potential only, Must be done ≤7 days prior to registration, and, if start of treatment is >7 days after initial testing, again ≤7 days 
prior to start of treatment. 
16Tumor measurement/breast imaging should be per institutional standard of care. Bi-dimensional measurement should be collected. Imaging reports from 
diagnosis and post-NAC should be submitted for all patients during pre-registration. 

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Parameter 
Pre-Reg 
Registration9 
(Baseline) 
Abemaciclib 
14-21 days 
prior to surgery 
Prior to 
surgery 
At time of 
surgery 
End of Study 
30-60 days 
after surgery10 
CORRELATIVE STUDIES: 
 
 
 
 
 
 
Research Tumor/Tissue Biopsy 
X 
 
 
 
X17 
 
Research tissue specimens from clinical procedures 
X18 
 
 
 
X 
 
Research blood specimens 
 
X 
 
X19 
 
 
Research stool specimens 
 
X20 
 
X21 
 
 
ADDITIONAL INFORMATION: 
 
 
 
 
 
 
Adverse Events/Symptom Assessment 
 
X 
 
X 
 
X 
Patient Medication Diary22 
 
 
X 
 
 
 
Submission of Surgical (Operative) and Pathology 
Reports 
X23 
 
 
 
X 
 
Note: Additional tests may be performed at the discretion of the treating investigator as clinically indicated. 
 
 
 
17 If the patient will not have surgery, a research biopsy should be performed 14-21 days after starting study drug. 
18 Collection of a representative block or slides from initial biopsy (prior to neoadjuvant chemotherapy) 
19 Collect research blood after completion of abemaciclib, prior to surgery 
20 Baseline stool collection kit may be given to patient any time after consent is obtained and pre-registration patient study ID is available. Preferred timeline is 
≤14 days prior to C1D1 
21 Pre-surgery stool kit can be collected up to 5 days prior to surgery 
22 NOTE: Study staff upload completed diary to Medidata Rave 
23 Submit reports from biopsy at the time of diagnosis as well as reports from Pre-Registration research biopsy for this study 

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7. TREATMENT PLAN 
Surgery should be planned/scheduled for no later than 12 weeks after the last dose of 
neoadjuvant chemotherapy. 
7.1 Biopsy during preregistration phase 
Breast biopsy to obtain tissue from residual tumor/tumor bed. Biopsies should be obtained 
from any residual abnormal tissue or if no tumor visible, biopsy around clip. If the 
research biopsy is adjacent to the existing clip and the clip is NOT removed, then no 
additional clip needs to be placed. 
If the research biopsy removes the existing clip, OR if the area biopsied is >1cm from the 
clip, then a clip should be placed to mark the area of the research biopsy. [In the situation 
where a second clip is placed, recommendation is to use a different shaped clip than the 
one already present in the breast]. 
NOTE: Ultrasound-guided biopsy obtain 6-8 cores using 14 gauge needle. 
If no target seen on ultrasound, then stereotactic biopsy should be performed 
obtaining 4-6 cores using 9 gauge needle. 
First and second core will be FFPE. The first core will be processed locally for H&E and 
used for eligibility. The H&E as well as the remaining portion of Core 1 and all of Core 2 
will be sent to Mayo Clinic in Rochester, MN, for research purposes.  
The remaining cores will be snap frozen (with the exception of Cores 5 and 6 at Mayo 
Rochester only which will be processed for PDX and organoids). Please see lab manual 
for specimen processing details. 
7.2 Cohort B Group 4: Abemaciclib Administration  
Agent administered orally twice daily for one cycle. 
Agent 
Dose Level 
Route 
Day 
ReRx 
abemaciclib 
150mg 
Oral (po) 
twice per day 
at least 
Days 1-14 
at most 
Days 1-21* 
NA 
*Length of treatment depends on when surgery is scheduled. Last dose should be taken 
one day prior to surgery. 
Abemaciclib cannot be started until ≥21 days after last dose of neoadjuvant 
chemotherapy, and ≥21 days after the last dose of pembrolizumab. 
If drug is stopped or delayed for any reason other than life threatening adverse event(s), 
patients may resume abemaciclib per protocol and may receive up to 42 total doses of 
drug prior to surgery. 

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7.3 Cohort B Group 4 Concomitant Treatment and Supportive Care Guidelines 
 Supportive care 
Patients should receive standard supportive care during treatment with abemaciclib. 
This treatment includes blood product support, antibiotic treatment, and treatment 
of other newly diagnosed or concurrent medical conditions.  
All blood products and concomitant medications such as antidiarrheals, analgesics, 
and/or antiemetics received from the first day of study treatment administration 
until 30 days after the final dose will be recorded in the medical records. 
 Diarrhea 
NOTE: For this study, patients will be given a prescription for loperamide at the 
same time as the abemaciclib is dispensed. 
In the event of diarrhea, supportive measures should be initiated as early as 
possible. These include the following: 
• 
At the first sign of loose stools, the patient should initiate anti-diarrheal 
therapy (e.g. loperamide) and notify the investigator for further instructions 
and appropriate followup.  
• 
Patients should also be advised to drink fluids (e.g., 8 to 10 glasses of clear 
liquids per day).  
• 
If diarrhea does not resolve with anti-diarrheal therapy within 24 hours to 
either baseline or Grade 1, then patient should contact study staff and dosing 
should be adjusted as outlined in Section 8.4.2. In the event of a weekend, 
patient will be instructed to contact physician on call. 
• 
Site personnel will assess response to adjusted anti-diarrheal therapy within 
24 hours. 
 Hepatic Monitoring 
Details for hepatic monitoring depend upon the severity and persistence of 
observed laboratory test abnormalities. To ensure patient safety the investigator 
should collect specific recommended clinical information and follow-up 
laboratory tests as shown in Appendix C: Lilly Guidance for Hepatic Treatment 
Emergent Abnormality. 
7.4 Duration of Therapy 
 Cohort B Group 3: Patients without residual disease  
Patients whose pre-registration biopsy specimen has no residual invasive 
disease identified on the H&E will proceed to surgery as scheduled. 
 Cohort B Group 4: Patients with residual invasive disease 
Patients who have residual invasive disease identified on H&E will receive 
abemaciclib for a minimum of 14 days and a maximum of 21 days such that the 
last dose of abemaciclib is administered the day prior to surgery. 
It is strongly recommended that the surgery date is scheduled and the date of 
initiation of abemaciclib calculated to be 15-22 days prior to date of surgery, 

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so that the patient receives a minimum of 14 days and a maximum of 21 days 
of treatment, with the goal that the last dose is administered the day prior to 
surgery. 
The reasons for discontinuation of abemaciclib include: 
• Treatment intolerability defined as:  
ANC <1,000/mm3, platelets <100,000/mm3, or any ≥Grade 2 treatment-
related adverse events 
• Intercurrent illness 
• Administration of alternative anti-cancer treatment 
• Request by patient to discontinue study treatment 
• Withdrawal of consent 
• Found to be ineligible after starting abemaciclib 
All reasons for discontinuation of therapy should be documented clearly in the 
medical record. 
If a subject discontinues treatment early and goes to surgery without receiving 
other anti-cancer therapy, surgical tissue/tumor and research blood will still be 
collected. 
7.5 Duration of Follow-Up  
1) Cohort B Group 3: Patients who undergo a pre-registration biopsy with no 
residual invasive disease identified on the H&E will proceed to surgery as 
scheduled. Surgical, imaging, and pathology reports will be submitted and 
patients will go off study after submission of reports. 
2) Cohort B Group 4: Patients who undergo a pre-registration biopsy and have 
residual invasive disease identified on the H&E, and who receive any amount of 
study drug will be followed for adverse events until approximately 30-60 days 
post-surgery. Submission of tumor tissue, and pathology and imaging reports 
should occur as outlined in Section 6. 
7.6 Criteria for Study Discontinuation 
 Study participation is complete upon withdrawal of consent. 
 Patients whose pre-registration biopsy has residual invasive disease identified 
on the H&E but refuse to start treatment with abemaciclib are considered a 
cancel. All onstudy materials and end of treatment case report form are to be 
completed. 
7.7 Treatment Following Surgery 
Following surgery, patients should proceed with standard guideline recommended 
adjuvant treatments.  
 
 

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8. EXPECTED ADVERSE EVENTS AND DOSING DELAYS/DOSE 
MODIFICATIONS –COHORT B GROUP 4 
8.1 Treatment Administration 
Abemaciclib will be administered orally at a dose of 150 mg (3 x 50mg tablets) twice 
daily for 14-21 days. 
Abemaciclib should be taken with food. (NOTE: Grapefruit and grapefruit juice should 
be avoided while taking abemaciclib.) 
If a patient misses a dose, she must be instructed not to make it up but just take her next 
regular dose.  
If a patient vomits any time after taking a dose, she must be instructed not to make it up 
but to resume the next regular dose. 
If a patient inadvertently takes an extra dose during a day, instruct patient to call her 
physician for instructions.  
CBC with diff is to be performed 3 days (±1 day) prior to surgery. Treatment with 
abemaciclib should be discontinued if ANC <1,000/mm3, platelets <100,000/mcL, or 
patients present with any ≥Grade 2 treatment-related adverse events. 
If a patient needs to stop treatment prematurely, she will have surgery on the scheduled 
date unless contraindicated (see Section 8.3.2). Surgery may be rescheduled at the 
discretion of the treating physician.  
Abemaciclib is predominantly cleared by oxidative metabolism via CYP3A4. Clinical 
drug interaction studies with a CYP3A inhibitor and CYP3A inducer significantly altered 
the PK of abemaciclib and its circulating major metabolites. Therefore, drugs that are 
strong and moderate inducers of CYP3A and/or strong inhibitors of CYP3A should be 
avoided or substituted if necessary. 
8.2 Anticipated Adverse Events 
The primary short term adverse events related to abemaciclib include neutropenia 
(Grade 1-3), diarrhea, nausea, and fatigue (Grade 1-2). Given the short term nature of this 
study, ≥Grade 3 adverse events are not anticipated. However, in the event these adverse 
events are observed, recommendations for management are included below. 
 Assessment of renal insufficiency 
Elevation of serum creatinine is observed with abemaciclib, and is due to a 
pharmacological inhibitory effect of abemaciclib on renal tubular transporters 
without affecting glomerular function. The rise in serum creatinine (mean increase, 
0.2 mg/dL) occurs within the first 28-days of abemaciclib, and and is reversible 
upon treatment discontinuation. Alternative markers (such as BUN, cystatin C level, 
or cystatin C calculated GFR), which are not based on creatinine, may be 
considered to determine whether renal function is impaired. 

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8.3 Management of Adverse Events 
 Diarrhea 
At enrollment, patients should receive instructions on the prompt management of 
diarrhea. Patients should be given a prescription for loperamide at the same time as 
the abemaciclib is dispensed. In the event of diarrhea, supportive measures should 
be initiated as early as possible. 
At the first sign of loose stools, the patient should initiate anti-diarrheal therapy, and 
notify the investigator for further instructions and appropriate follow up. If diarrhea 
does not resolve with anti-diarrheal therapy within 24 hours to either baseline or 
Grade 1, then patient should contact study staff and dose adjustments should be 
made as outlined in Section 8.0. Patients should be advised to drink fluids (e.g. 8 to 
10 glasses of clear liquids per day).  
Diarrhea could be managed conservatively with loperamide. The recommended 
dose of loperamide is 4 mg at first onset, followed by 2 mg every 2-4 hours until 
diarrhea free (maximum 16 mg/day). 
In the event of Grade 3 or 4 diarrhea, the following supportive measures are 
allowed: hydration, octreotide, and antidiarrheals.  
If diarrhea is severe (requiring intravenous rehydration) and/or associated with 
fever or severe neutropenia (Grade 3 or 4), broad-spectrum antibiotics must be 
prescribed. Patients with severe diarrhea or any diarrhea associated with severe 
nausea or vomiting should be hospitalized for intravenous hydration and correction 
of electrolyte imbalances. 
 Other Adverse Events 
Any Grade 3 or 4 adverse event, regardless of attribution, should result in drug 
discontinuation. Surgery should be postponed until all adverse events are resolved 
to <Grade 3. 
Note: Surgery in the setting of ≤Grade 2 neutropenia is allowed, given that 
neutrophil recovery is rapid after stopping abemaciclib (see Investigator Brochure). 
8.4 Dose Modifications/ Delays 
 Dose Adjustments for Abemaciclib 
Dose 
Level 
Abemaciclib 
0* 
150 mg every 12 hours 
-1 
100 mg every 12 hours 
-2 
50 mg every 12 hours 
*Starting dose level 
Study drug must be discontinued if further dose reduction is required 
beyond 50mg every 12 hours. 

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Study drug should be discontinued if adverse events of ≥Grade 2 do not resolve 
within 7 days of onset. 
If study drug is omitted for >7 days, then permanently discontinue study drug. 
NOTE: Specific dose modifications/delays are in the subsections below. 
 
 Dose Modifications for Diarrhea 
CTCAE Grade 
Abemaciclib Dose Modifications 
Grade 1 
No dose modification is required.  
Grade 2  
If AE does not resolve within 24 hours to ≤Grade 1, omit dose 
for up to 7days.  
Dose reduction is not required. 
Grade 2 that persists or recurs after 
resuming the same dose despite 
maximal supportive measures  
Omit dose until AE resolves to ≤Grade 1 for up to 7 days 
Resume at next lower dose 
Grade 3 or 4 or requires 
hospitalization 
 
 Dose Modifications for Increased Alanine Aminotransferase (ALT) and 
Aspartate Aminotransferase (AST) 
CTCAE Grade 
Abemaciclib Dose Modifications 
Grade 1 (>ULN-3.0 x ULN) 
Grade 2 (>3.0-5.0 x ULN) 
No dose modification is required. 
Persistent or Recurrent Grade 2, or Grade 3 
(>5.0-20.0 x ULN) that does not resolve with 
maximal supportive measures within 7 days to 
baseline or Grade 1 
Omit dose until AE resolves to baseline or Grade 1 
for up to 7 days. 
Resume at next lower dose. 
≥Grade 2 (>3.0 x ULN) with total bilirubin >2 x 
ULN, in the absence of cholestasis 
Discontinue abemaciclib 
Grade 4 (>20.0 x ULN) 
Discontinue abemaciclib. 
To ensure patient safety the investigator should collect specific recommended clinical 
information and follow-up laboratory tests as shown in Appendix C.  
 
 Dose Modifications for Infection, Bleeding, Anemia, Neutropenia and Other 
Cytopenias 
CTCAE Grade 
Abemaciclib Dose Modifications 
Grade 1 or 2 
No dose modification is required. 
Grade 3 
Omit dose until AE resolves to ≤Grade 2. 
Dose reduction is not required. 
Grade 3, recurrent, or Grade 4 
Omit dose until AE resolves to ≤Grade 2. 
Resume at next lower dose. 
Patient requires administration of a 
blood cell growth factor 
Omit abemaciclib dose for at least 48 hours after the last dose of 
blood cell growth factor and until AE resolves to ≤Grade 2. 
Resume abemaciclib at next lower dose unless the dose was already 
reduced for the AE that led to the use of the growth factor. 
 

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 General Guidance for Increases in Serum Creatinine and Assessment of 
Renal Insufficiency 
Abemaciclib has been shown to increase serum creatinine due to inhibition of renal 
tubular transporters without affecting glomerular function (as measured by iohexol 
clearance). In clinical studies, increases in serum creatinine occurred within the first 
month of abemaciclib dosing, remained elevated but stable through the treatment 
period, were reversible upon treatment discontinuation, and were not accompanied by 
changes in markers of renal function, such as blood urea nitrogen (BUN), cystatin C, 
or calculated glomerular filtration rate based on cystatin C. 
 
 Dose Modifications for Interstitial Lung Disease (ILD)/Pneumonitis 
CTCAE Grade 
Abemaciclib Dose Modifications 
Grade 1 or 2 
No dose modification is required. 
Persistent or recurrent Grade 2 
toxicity that does not resolve with 
maximal supportive measures 
within 7 days to baseline or Grade 
1 
Omit dose until toxicity resolves to baseline or Grade 1. 
Resume at next lower dose. 
Grade 3 or 4 
Discontinue abemaciclib. 
 
 General Guidance for Venous Thromboembolic Events 
VTE has been identified as an adverse drug reaction (ADR) for abemaciclib in 
combination with endocrine therapy. However, in studies with single-agent 
abemaciclib use in the metastatic breast cancer or other tumor types, including 
NSCLC, no increased rates of VTEs were observed as compared to the incidence of 
VTEs for these particular patient populations who were treated with other anticancer 
agents. At this time, the mechanism underlying the association between abemaciclib 
and the occurrence of VTEs is not known. Venous thromboembolic events have been 
reported with other CDK4 and 6 inhibitors, and ET is known to be associated with the 
occurrence of VTEs. Monitor patients for signs and symptoms of deep vein 
thrombosis and pulmonary embolism and treat as medically appropriate. 
 
 Dose Modifications for Venous Thromboembolic Events 
CTCAE Grade 
Abemaciclib Dose Modifications 
Grade 1 or 2 
No dose modification is required. 
Grade 3 or 4 
Suspend dose and treat as clinically indicated. Abemaciclib 
may be resumed when the patient is clinically stable. 
 
 
 

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 Dose Modifications for Other Adverse Events Not Specified in Above Tables 
CTCAE Grade 
Abemaciclib Dose Modifications 
Grade 1 or 2 
No dose modification is required. 
Persistent or recurrent Grade 2 AE 
that does not resolve with maximal 
supportive measures within 7 days 
to baseline or Grade 1 
Omit dose until AE resolves to baseline or Grade 1 for up to 
7 days. 
Resume at next lower dose. 
Grade 3 or 4 
 
 Guidelines for Pregnancy and Nursing 
Abemaciclib can cause fetal harm when administered to a pregnant person. In animal 
studies, abemaciclib was teratogenic and caused decreased fetal weight at maternal 
exposures that were similar to human clinical exposure based on the area under the 
plasma concentration versus time curve (AUC) at the recommended human dose. 
Therefore, teratogenicity is considered an important potential risk for abemaciclib. 
There are no available human data informing the drug-associated risk. Advise 
pregnant persons of the potential risk to a fetus. Additionally, there are no available 
data on effects of breastfeeding. Advise a nursing person to discontinue breastfeeding 
during treatment with abemaciclib. 
• Persons of childbearing potential must have a negative serum pregnancy test 
within 7 days of the first dose of abemaciclib and agree to use a highly effective 
contraception method during the treatment period and for 3 weeks following the 
last dose of abemaciclib.  
• Contraceptive methods may include an intrauterine device [IUD] or barrier 
method. If condoms are used as a barrier method, a spermicidal agent should be 
added as a double barrier protection. 
• Cases of pregnancy that occur during maternal exposures to abemaciclib should 
be reported. If a patient is discovered to be pregnant following abemaciclib 
initiation, that person must discontinue treatment immediately. Data on fetal 
outcome and breast-feeding are to be collected for regulatory reporting and drug 
safety evaluation. 

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9. DRUG FORMULATION/STORAGE/SUPPLY 
9.1 Abemacliclib (Verzenio®, LY2835219, NSC 783671) 
Abemaciclib is an investigational agent and will be supplied free-of-charge from Eli 
Lilly and Company.  
 Background:  
Abemaciclib is a selective and potent small-molecule CDK4 and CDK6 dual 
inhibitor. 
 Formulation: 
Abemaciclib will be provided as 50 mg tablets. 
 Preparation and storage 
Abemaciclib must be stored at room temperature. For specific storage instructions 
refer to the product label. 
 Administration 
Take abemaciclib with food at approximately the same times every day. Swallow 
tablets whole and do not chew, crush, or split tablets before swallowing. Do not take 
tablets if broken, cracked, or otherwise not intact. If a patient misses or vomits a dose, 
take the next dose at its scheduled time. 
 Pharmacokinetic information 
 Absorption 
Abemaciclib absorption is slow, with a median Tmax of 8.0 hours. In the 
therapeutic dose range of 50-200 mg, the increase in plasma exposure (AUC) and 
Cmax is dose proportional. Steady state was achieved within 5 days following 
repeated BID dosing, and abemaciclib accumulated with a geometric mean 
accumulation ratio of 3.3 (80% CV) and 4.6 (88% CV) based on Cmax and 
AUC, respectively. 
 Bioavailability 
The absolute bioavailability of abemaciclib is 45% (90% CI: 40-51%). 
 Distribution 
Abemaciclib was highly bound to plasma proteins in humans (mean bound 
fraction was approximately 96-98%), and the binding was independent of 
concentration from 152 to 5066 ng/mL. Abemaciclib binds to both human serum 
albumin and alpha-1-acid glycoprotein. The geometric mean systemic volume of 
distribution is approximately 747 L (68.6% CV). In patients with advanced 
cancer, concentrations of abemaciclib and its active metabolites M2 and M20 in 
cerebrospinal fluid are comparable to unbound plasma concentrations. 
 Metabolism 
Hepatic metabolism is the main route of clearance for abemaciclib. Abemaciclib 
is metabolized to several metabolites primarily by CYP3A, with formation of N-
desethyl abemaciclib (M2) representing the major metabolism pathway. 

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Additional metabolites include hydroxyabemaciclib (M20), hydroxy-N-
desethylabemaciclib (M18), and an oxidative metabolite (M1). Metabolites N-
desethylabemaciclib (M2) and hydroxyabemaciclib (M20) are active with similar 
potency as abemaciclib. 
 Half-life elimination 
The geometric mean hepatic clearance (CL) of abemaciclib was 21.8 L/hours 
(39.8% CV), and the mean plasma elimination half-life for abemaciclib in 
patients was 24.8 hours (52.1% CV). 
 Excretion 
After a single oral dose of [14C]-abemaciclib, approximately 81% of the dose 
was excreted in feces and 3.4% excreted in urine. The majority of the dose 
eliminated in feces were metabolites. 
 Potential Drug Interactions 
Abemaciclib is predominantly cleared by oxidative metabolism via CYP3A. 
Clinical drug interaction studies with a CYP3A inhibitor and CYP3A inducer 
significantly altered the PK of abemaciclib and its circulating major metabolites.  
CYP3A inducers 
Avoid concomitant use of CYP3A inducers and consider alternative agents. 
CYP3A inhibitors 
Avoid concomitant use of strong CYP3A inhibitors (for example, voriconazole) 
and use caution with coadministered moderate (for example, ciprofloxacin) or 
weak (for example, ranitidine) CYP3A inhibitors. If coadministration with a 
strong CYP3A inhibitor is unavoidable, reduce the abemaciclib dose to 100 mg 
twice daily or, in the case of ketoconazole, reduce the abemaciclib dose to 50 mg 
twice daily. In patients who have had a dose reduction to 100 mg twice daily due 
to adverse reactions, further reduce the abemaciclib dose to 50 mg twice daily. 
Avoid grapefruit or grapefruit juice. If a CYP3A inhibitor is discontinued, 
increase the abemaciclib dose (after 3-5 half-lives of the inhibitor) to the dose that 
was used before starting the inhibitor.  
 Known potential adverse events 
Most common adverse reactions (incidence ≥20%) were diarrhea, neutropenia, 
nausea, abdominal pain, infections, fatigue, anemia, leukopenia, decreased 
appetite, vomiting, headache, and thrombocytopenia. 
In the MONARCH 2 study, venous thromboembolisms (VTEs) were experienced 
by 21 patients (4.8%) in the abemaciclib plus fulvestrant arm and by 2 patients 
(0.9%) in the placebo plus fulvestrant arm. In the MONARCH 3 study, VTEs 
were experienced by 16 patients (4.9%) in the abemaciclib plus non-steroidal 
aromatase inhibitor (NSAI) arm and by 1 patient (0.6%) in the placebo plus NSAI 
arm. In these studies, risk factors for VTEs were balanced between the study 
arms, and overall there was no specific risk factor identified that could be 
predictive of VTEs on treatment with abemaciclib. 

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 Drug Ordering and Accountability 
Abemaciclib will be provided free of charge by Eli Lilly & Co. 
Each institution will order the drug through drug supplier. (See Pharmacy Manual 
for order form.) 
NOTE: Local drug destruction may occur if the site provides a copy of their local 
drug destruction policy to the sponsor (Mayo Clinic) and the respective drug 
supplier (Eli Lilly & Co). 
 
10. CORRELATIVE/SPECIAL STUDIES  
10.1 Collection Tables 
 Cohort B Group 3 
Specimen Type 
Pre-Reg24 
After Registration 
prior to treatment25 
(Baseline) 
At time of 
surgery 
Research Blood  
(70 mL) 
 
X 
 
Research Stool 
 
X 
 
Research Tissue 
X  
(needle cores from 
biopsy and  
unstained slides or tissue 
blocks from archived 
FFPE tissue if available) 
 
X  
(FFPE tissue 
block/slides 
required) 
See Lab Manual for specifics of specimen collection and processing 
Note: Surgical FFPE tissue block/slides (with residual tumor or tumor bed) is 
required at time of surgery to assess study endpoints.  
Note: If patient does not have surgery, then a research biopsy should be 
performed and needle core biopsies obtained – follow same procedures as for Pre-
Registration biopsy, although an additional clip does not need to be placed. 
 
 
 
24 ≤35 days prior to registration (after completion of NAC) 
25 Baseline stool kit may be collected any time after consent is obtained and pre-registration patient study ID is 
available. Preferably ≤14 days prior to first treatment with abemaciclib 

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 Cohort B Group 4 
Specimen Type 
Pre-Reg26 
After 
Registration 
prior to 
treatment 
(Baseline) 
Prior to 
surgery  
At time of 
surgery 
Research Blood 
(70 mL) 
 
X27 
X 
 
Research Stool 
 
X28 
X 
 
Research Tissue 
X  
(needle cores from 
biopsy and  
unstained slides or 
tissue blocks from 
archived FFPE tissue 
if available) 
 
 
X  
(FFPE 
tissue 
block/slides 
required) 
See Lab Manual for specifics of specimen collection and processing 
Note: Surgical FFPE tissue block/slides (with residual tumor or tumor bed) is 
required at time of surgery to assess study endpoints.  
Note: If patient does not have surgery but has received at least 14 days of 
treatment, then a research biopsy must be performed and needle core biopsies 
obtained – follow same procedures as for Pre-Registration biopsy, although an 
additional clip does not need to be placed. 
 
 
 
26 ≤35 days prior to registration (after completion of NAC) 
27 ≤14 days prior to first treatment 
28 Baseline stool collection kit may be given to patient any time after consent is obtained and pre-registration patient 
study ID is available. Preferably ≤14 days prior to first treatment with abemaciclib. 

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10.2 Blood Samples 
 Circulating Tumor Cells (CTC) 
Whole blood samples will be collected to isolate circulating tumor cells. These 
samples will be shipped immediately according to the lab manual to Mayo Clinic 
Rochester for processing. 
The markers used for CTC identification may include DAPI or SYTOX orange, 
cytokeratin, EpCAM, ER, HER2, ALDH1, and CD44 to distinguish between 
epithelial-like CTCs and EMT/stem-cell like CTCs. All CTCs will be assessed for 
vimentin expression. CD45 will be used to distinguish WBCs (CD45 positive) from 
CTCs (CD45 negative). CTC populations will be isolated for DNA extraction using 
established methods to allow for mutation analysis. See Lab Manual for collection 
and processing instructions 
It is known that CTCs have biologic relevance in epithelial malignancies, and their 
relative abundance in the peripheral blood has strong correlations with disease 
related outcomes in early stage and advanced breast cancer50-54. Beyond simple 
enumeration, molecular profiling of these cells may provide clinically relevant 
predictive information to guide the selection of therapy. Deriving detailed molecular 
signatures for isolated CTCs may also facilitate drug discovery by assessing the 
markers unique to progression, such that relapsing patients may be treated for their 
current molecular disease. 
The Mayo group has established experience detecting and isolated purified CTCs 
from breast cancer patients, as well as the capability to design 4- or 6-marker panels 
to capture specific phenotypic populations of CTCs.  
 Cell-free DNA (cfDNA) 
Whole blood samples will be collected to isolate cfDNA. These specimens will be 
shipped immediately to Mayo Clinic Rochester according to the Lab Manual.  
Samples will be stored until we are ready for DNA extraction and mutational 
analysis. Selected mutations identified at baseline will be verified and quantified in 
all cfDNA samples. 
 Pharmacogenomics 
Whole blood sample will be collected for DNA and proteomics. These specimens 
will be shipped immediately to Mayo Clinic Rochester according to the Lab Manual.  
Pharmacogenomics is the study of the role of genetic variation in the individual 
variation of drug response and toxicity. In the specialty of oncology, drug response 
phenotypes vary based upon both tumor (somatic), as well as host (germ-line) genetic 
variation. Our aims are to identify single nucleotide polymorphisms (SNPs) or 
genetic variants associated with treatment resistance and development of adverse 
drug effects. DNA will be genotyped (or sequenced) based on the most up to date 
technology. Current technology platforms such as the Illumina OmniExpress 
BeadChip (San Diego, CA, USA) are often used for GWAS studies but newer 
platforms will be used at the time of genotyping/sequencing. Sample quality control 
will be carried out by methods including identity-by-state to evaluate cryptic 

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relatedness for each sample and population stratification by the use of principal 
component analysis to exclude genetically heterogeneous samples from further 
analysis. 
 Peripheral blood immune phenotype analyses with CyTOF 
Whole blood sample will be collected for PMBCs according to the lab manual. 
Analyses of tumor-host immune interaction studies have largely focused on 
exploring the interplay between cancer cells and immune cells at the tumor tissue 
level. Analyses evaluating the tumor microenvironment have identified that the 
robustness of immune cell infiltration in TNBC is both prognostic and predictive of 
response to chemotherapy and immunotherapy. We will aim to evaluate whether 
such changes in CD8+ T cells, regulatory T cells, and other immune cell populations 
are detectable beyond the tumor microenvironment, in peripheral blood. To this end, 
we will leverage multiplexed technologies such as Cytometry by Time-of-Flight 
(CyTOFTM), which have greatly improved the ability to evaluate a large number of 
surface markers at the single cell level, allowing for deep characterization of 
circulating immune cell populations in peripheral blood specimens.  
Here, we will test the hypothesis that treatment with abemaciclib will be associated 
with expansion of the peripheral blood CD8+ T cell compartment, and contraction 
of the peripheral blood Treg compartment, mirroring what has been observed in 
tissues preclinically.  
10.3 Stool Samples 
 Research Stool Collection 
Timepoints: 
• Pre-Registration –stool kit provided at pre-registration (All patients) 
• Prior to surgery–stool kit provided prior to surgery Cohort B Group 4 only) 
Samples will be collected and processed per lab manual and used for multi-omic 
analysis to evaluate changes in the microbiome with exposure to abemaciclib. 
There is growing evidence that the large number of residential microbes (e.g. viruses, 
bacteria, eukaryotes, others) that colonize the human gastrointestinal tract have 
fundamental roles in health and disease (Kwa M, 2016). It has recently emerged that 
the gastrointestinal microbiome can influence cancer development and therapeutic 
response. For example, studies have shown that the composition of gut bacteria can 
influence the therapeutic efficacy of immune checkpoint inhibitors, and that 
therapeutic efficacy can be improved through its modulation (Gopalakrishnan V, 
2018) (Routy B, 2018). Further, there is evidence that variations in the composition 
and activities of enteric bacterial genes that metabolize estrogen may influence the 
development of postmenopausal HR-positive breast cancer. The development of a 
criterion based not only on the genetic makeup of a tumor biopsy, but also on the gut 
microbiome and other biomolecular features (e.g. serum metabolites, cytokine 
profiling) that can determine whether a particular drug may be effective has garnered 
interest in personalized medicine (Chen R, 2013). 

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10.4 Tissue Samples 
 Research Biopsy Collection  
Timepoints: 
• with clip placement during Pre-Registration (all patients); and 
• End of abemaciclib, if surgery will not be performed (Cohort A Group 2 
and Cohort B Group 4 only) 
Ultrasound-guided biopsy to obtain 6-8 tissue cores using 14 gauge needle. 
If no target seen on ultrasound, then stereotactic biopsy should be performed 
obtaining 4-6 tissue cores using 9 gauge needle. 
First and second tissue cores will be submitted for routine FFPE processing. 
A section from each of the first two cores will be evaluated by H&E for patient 
eligibility. The H&Es, corresponding FFPE tissue blocks (with residual core 1 
tissue and residual core 2 tissue) will be sent to Mayo Clinic in Rochester, MN, for 
research purposes.  
The remaining tissue cores will be snap frozen (with the exception of cores 5 and 6 
at Mayo Rochester only which will be processed for PDX). Please see Lab Manual 
for specimen processing details and core prioritization. 
Additional FFPE derived tissue sections will be used to evaluate the effects of 
abemaciclib on the immune phenotype of FFPE tumor sections will be processed to 
examine the following: 
• genes directly involved in tumor cell antigen presentation (e.g., B2M, HLA-
A, HLA-B, HLA-C, TAP1, TAP2, TAPBP) 
• interferon-stimulated genes (ISGs) that regulate antigen presentation (e.g., 
STAT1, NLRC5) and other ISGs (e.g., IRFs, OAS2) 
• genes involved in dsRNA response (e.g., DDX58, DHX58) 
• genes encoding interferons, including type 3 IFNs (e.g., IFNL1, IFNL2, 
IFNL3) 
• genes indicating a cytotoxic T cell response (e.g., PRF1, GZMB) 
• Treg-specific transcription factor genes (e.g., FOXP3, IKZF2). 
DNA and RNA extraction will be performed and sequencing will be completed.  
Baseline research biopsy, will require hematoxylin and eosin (H & E) staining to be 
performed on the first and second cores obtained as part of standard of care 
procedure. Additional analyses will be performed on remaining tissue at a later date. 
 Surgical Tissue Collection 
At the time of surgery the surgical specimen will be processed according to Mayo 
Clinic Pathology Lab standards. The pathologist should ensure the identification of 
the appropriate clip denoting the research biopsy site, and collect and submit residual 
tumoral tissue or tumor bed around the research biopsy clip to ensure evaluation of 
tissue in the same region as the research biopsy. The surgical tissue will be prioritized 
according to research needs or best utilization as indicated in the lab manual. 

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In cases where there is no residual invasive cancer in the breast, a sample of the 
residual tumor bed adjacent to the research biopsy site (denoted by a clip – as above) 
should be submitted according to the lab manual. 
In cases with residual disease in the lymph nodes (sentinel lymph node and/or any 
other axillary lymph nodes), then tissue from grossly positive lymph nodes will be 
flash frozen. 
In the case where there is multifocal disease remaining in the breast at the time of 
surgery tissues from each of the residual tumors will be submitted for processing 
(following the above protocol). 
For patients with bilateral disease the tissue from the index side will be processed as 
outlined above and any residual disease from the contralateral side will be processed 
per usual pathology guidelines and not submitted. 
 PDX (Mayo Clinic Rochester Patients Only) 
Xenografts will be generated in Dr. Liewei Wang’s lab (Mayo Clinic). Her lab has 
already successfully generated a series of breast cancer models for all subtypes from 
both primary breast cancer as well as metastatic breast cancer using core biopsy or 
surgical samples. In this study, to generate individual tumor xenograft line for drug 
screening, drug cytotoxicity assay, specimen obtained during the biopsy will be 
immediately injected into NGS mice for the creation of human tissue xenografts (see 
IACUC A49111 and IACUC A00003279-17). The xenograft tumor samples will be 
used to determine the functional implications of tumor alterations identified from 
tumor sequencing and for future drug screening and cytotoxicity assay. These lines 
will be also useful for future research purposes, such as testing novel compounds and 
different regimens. All the tumor tissues from xenografts will be stored on Gonda 19-
466E, Mayo Clinic, 200 First St SW, Rochester MN 55905. 
 Organoids (all sites) 
Organoids will be generated in Dr. Liewei Wang’s lab (Mayo Clinic, Rochester, MN) 
 Drug Screen (ex vivo) 
Tumor tissue will be sent for drug screening using a 3D microcancer model to 
identify potential targets which could also be validated in the PDX created at 
progression. Results will be interpreted in the context of sequencing data from ctDNA 
and/or tissue collected after abemaciclib administration. 
 Microcancers: 3D models of solid tumors for drug sensitivity screening 
Validation of companion therapeutic biomarkers for personalized therapy of solid 
tumors has been slow because of the lack of appropriate model systems to test 
biomarker-guided therapies. In the past, conventional cell lines have been used 
extensively with little success. It is now believed that their forced adaptation to two-
dimensional (2D) culture conditions leads to loss of key genetic and functional 
characteristics and gain of others, making them unreliable models of human cancer. 
Conversely, genetically engineered animal cancer models rarely recapitulate human 
cancer in a personalized manner, while models based on human cancer xenografts in 
immunocompromised mice take months to develop. The advent of three-dimensional 

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(3D) tissue modeling in vitro, is a promising mechanism to fill the gap and to provide 
reliable, sensitive, and affordable characterization of tumor samples for biomarker 
validation and response to treatment.  
Multiple studies have shown that human cancer cells extracted fresh from solid 
tumors can grow in mice or in 3D culture in a superior manner to growth in 2D 
culture for drug sensitivity studies. Likely explanations for this are the presence of 
both cancer cells and tumor microenvironment in a more physiological 3D context, 
and the overreliance of cells grown in 2D culture to tension-induced signaling, 
otherwise known as mechanotransduction, for their growth in plastic. Interestingly, 
growth of normal human tissues in 3D culture has been achieved for many tissues, 
under conditions that preserve the stem cell compartment and retain the normal tissue 
architecture.  
3D model systems have several advantages over animal models. A special 
consideration is that all proliferative models of drug screening, including 2D, 3D 
organoids, and PDX models, are subject to selective pressure and tumor evolution. 
The tumor microenvironment is increasingly recognized as a key contributor to 
cancer progression and resistance to therapy. Therefore, removing selective pressure 
by optimizing culture conditions, and maintaining the tumor microenvironment are 
essential for accurate prediction of response to treatment.  
The laboratory of Dr. Panagiotis Anastasiadis has developed a non-proliferative 3D 
microcancer model process that minimizes selective pressure and maintains the 
microenvironment. This model provides the ability to maintain individual patients’ 
tumors in short-term 3D culture, in a manner that allows both biomarker validation 
and drug screening in a timely and sensitive manner. The Anastasiadis lab has 
developed and optimized the process and has successfully grown every patient tissue 
received to date, including tumors of the pancreas, liver, breast, brain, kidney, ovary 
and bladder. They have optimized 96-well based cytotoxicity assays and established 
assay sensitivity to as few as 500 cells, allowing the rapid interrogation of potential 
biomarkers and drug treatments in single patient biopsies.  
Dr. Liewei Wang will utilize Dr. Anastasiadis’ established methods. 
The drug screening panel will include FDA approved regimens selected for 
BEAUTY-2 (MC1734). 
 JAK2 
Triple negative breast cancer (TNBC) is an aggressive disease with poor clinical 
outcomes. Thus there is a need to identify markers that can be exploited for improved 
clinical care. We and subsequently others have identified amplification of 
chromosome 9p24.1 encoding PD-L1, PD-L2, and JAK2 (the PDJ amplicon) as a 
clinically relevant driver of aggressive TNBC. Furthermore there is emerging data 
suggesting that the presence of the amplicon is associated with increased 
JAK2/pSTA3 signaling and a reprogrammed immune environment. Recent studies 
have demonstrated that presence of PDJ amplifications is associated with response to 
checkpoint inhibitors (ICI). Studies have shown that PDJ can be induced with 
chemotherapy. In order to determine whether this is a biomarker of immune 

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dysregulation and an inducible biomarker we will perform JAK2 FISH analysis on 
the pretreatment biopsies and biopsies on patients with residual disease. The 
requirement will be 2 unstained slides (5 um sections) with one scanned H&E slide 
with the area of interested circled. Analysis of JAK2 amplification will be performed 
in the Immunohistochemistry Core at the Mayo Clinic in Rochester, MN. 
10.5 Genetic Testing 
Participants will be given information as part of the informed consent process that samples 
will be used for research tests that will include genetic studies and testing. The intent is not 
to give participants (or his/her medical providers) the results of any testing done for 
research purposes; however, incidental germline (heritable) mutations may be identified of 
which a participant may or may not already be aware. In the case that an incidental genetic 
finding is identified, the Protocol Chair of this project will be notified. The possible 
decisions for handling incidental findings may include notification of the participant (and 
provider); recommendation for genetic counseling, which may or may not include genetic 
testing (e.g., if the finding was not done in a CLIA certified laboratory); or, neither. In 
general, a member of the participant’s treating team will be given the information to help 
with notification. In all cases, the current policy of the Mayo Clinic and local/participating 
site IRB, as applicable, will be followed and any additional approvals that may be required 
prior to participant notification will be secured in advance. 
10.6 Additional Information 
Submission of data for Genome Wide Association Studies (GWAS) is not currently 
planned; however, subjects will be asked for permission in the informed consent process. 
A revision to the protocol and/or any regulatory approvals will be secured prior to any 
GWAS submission or inclusions in the future, if applicable.  
11. SPECIMEN BANKING 
The study Protocol Chair and collaborators have approval to use all research biospecimens 
collected during the conduct of this trial to address the research questions described in the 
protocol document. All future use of residual or repository specimens collected in this trial 
for purposes not prospectively defined will require review and approval by Mayo Clinic 
Cancer Center according to its established policies, whether the specimens are stored in a 
central site or at a local institution in a virtual repository. 
Secondary use of biospecimens for new endpoints must be submitted to the study chairs for 
possible reviewe. 
12. MEASUREMENT OF EFFECT: COHORT A GROUP 2 AND COHORT B 
GROUP 4 
CD8/FOXP3 protein expression will be determined in tumor tissue obtained by image 
guided percutaneous breast biopsy after completion of NAC (prior to abemaciclib) and in 
residual invasive breast cancer obtained at surgery or post-treatment biopsy (after 
completion of 14-21 days of abemaciclib).  

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13. ADVERSE EVENT REPORTING REQUIREMENTS 
13.1 General 
Adverse event collection and reporting is a routine part of every clinical trial. This study 
will use the descriptions and grading scales found in the NCI Common Terminology 
Criteria for Adverse Events current version (CTCAE v5.0) that is available at 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
Information on all adverse events, whether reported by the participant, directly observed, 
or detected by physical examination, laboratory test or other means, will be collected, 
recorded, followed and reported as described in the following sections.  
Adverse events experienced by participants will be collected and reported from initiation 
of study medication, throughout the study, and within 30 days of the last dose of study 
medication. Participants who experience an ongoing adverse event related to a study 
procedure and/or study medication beyond 30 days will continue to be contacted by a 
member of the study team until the event is resolved, stabilized, or determined to be 
irreversible by the participating investigator.  
Participants should be instructed to report any serious post-study event(s) that might 
reasonably be related to participation in this study. The investigator should notify the IRB 
and any other applicable regulatory agency of any unanticipated death or adverse event 
occurring after a participant has discontinued or terminated study participation that may 
reasonably be related to the study.  
NOTE: All sites must notify Study Chair within twenty-four (24) hours of Investigator 
and/or Institution receiving notification of any “serious” adverse event experienced by a 
patient participating in the Study and receiving Study Drug. Study Chair will review and 
provide approval to forward to Lilly within 2 days of notification. 
Investigator and Institution further agree to make available promptly to Lilly such records 
as may be necessary and pertinent for Lilly to further investigate an adverse event in the 
Study that is possibly associated with the Study Drug. 
13.2 Definitions 
 Adverse Event (AE) 
An adverse event is any undesirable sign, symptom or medical condition or experience 
that develops or worsens in severity after starting the first dose of study treatment or 
any procedure specified in the protocol, even if the event is not considered to be related 
to the study.  
Abnormal laboratory values or diagnostic test results constitute adverse events only if 
they induce clinical signs or symptoms or require treatment or further diagnostic tests.  
 Serious adverse event (SAE) 
A serious adverse event is an undesirable sign, symptom, or medical condition which: 
• 
is fatal; 
• 
is life-threatening; 
• 
requires or prolongs inpatient hospitalization for ≥24 hours; 

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• 
results in persistent or significant disability/incapacity to conduct normal life 
functions; 
• 
constitutes a congenital anomaly or birth defect; or 
• 
jeopardizes the participant and requires medical or surgical intervention to prevent 
one of the outcomes listed above; 
• 
Important Medical Events (IME) that may not result in death, be life threatening, 
or require hospitalization may be considered serious when, based upon medical 
judgment, they may jeopardize the patient or subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
Events not considered to be serious adverse events are hospitalizations for: 
• 
routine treatment or monitoring of the studied indication, not associated with any 
deterioration in condition, or for elective procedures 
• 
elective or pre-planned treatment for a pre-existing condition that did not worsen, 
or that is required per protocol 
• 
emergency outpatient treatment for an event not fulfilling the serious criteria 
outlined above and not resulting in inpatient admission 
• 
respite care 
• 
Death due to disease progression unless attributable to the study drug(s) 
• 
A hospitalization due to an expected adverse event (e.g., hospitalization due to 
expected febrile neutropenia). 
 Expectedness  
• 
Expected: Expected adverse events are those that have been previously identified 
as resulting from administration of the agent. For the purposes of this study, an 
adverse event is considered expected when it appears in the current adverse event 
list, the Investigator’s Brochure, the package insert or is included in the informed 
consent document as a potential risk. 
• 
Unexpected: An adverse event is considered unexpected when it varies in nature, 
intensity or frequency from information provided in the current adverse event list, 
the Investigator’s Brochure, the package insert or when it is not included in the 
informed consent document as a potential risk 
 Attribution 
Attribution is the relationship between an adverse event or serious adverse event and 
the study treatment. Attribution will be assigned as follows: 
• 
Definite – The AE is clearly related to the study treatment. 
• 
Probable – The AE is likely related to the study treatment. 
• 
Possible – The AE may be related to the study treatment.  
• 
Unlikely - The AE is doubtfully related to the study treatment. 
• 
Unrelated - The AE is clearly NOT related to the study treatment. 
13.3 Reporting Procedures 
All adverse events will be captured on the appropriate study-specific case report forms 
(CRFs). 
 

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 Summary of Event Reporting for this Trial 
WHO: 
WHAT form: 
WHERE to send: 
Mayo 
Clinic Sites 
Mayo Clinic Cancer Center SAE Reporting Form: 
http://livecycle2.mayo.edu/workspace/?startEndpoint=MC4
158-56/Processes/MC4158-56-Process.MC4158-56  
AND attach 
Pregnancy Reporting 
http://ctep.cancer.gov/protocolDevelopment/electronic_a
pplications/docs/PregnancyReportFormUpdated.pdf  
Send a copy to Lilly: 
Fax 866-644-1697 or: 317-453-3402 
NOTE: MCCC form will automatically 
be sent to 
CANCERCROSAFETYIN@mayo.edu 
and RSTP2CSAES@mayo.edu  
Mayo 
Clinic Sites 
Mayo Clinic Cancer Center SAE Reporting Form: 
http://livecycle2.mayo.edu/workspace/?startEndpoint=MC4
158-56/Processes/MC4158-56-Process.MC4158-56  
AND attach 
MedWatch 3500A: 
http://www.fda.gov/downloads/AboutFDA/ReportsManuals
Forms/Forms/UCM048334.pdf  
Submit a copy to Lilly: 
Fax 866-644-1697 or: 317-453-
3402 
NOTE: MCCC form will automatically 
be sent to 
CANCERCROSAFETYIN@mayo.edu 
and RSTP2CSAES@mayo.edu 
 EXPECTED Serious Adverse Events: Protocol Specific Exceptions to 
Expedited Reporting 
For this protocol only, the following Adverse Events/Grades are expected to 
occur within this population and do not require Expedited Reporting. These 
events must still be reported via Routine Reporting (see Section 13.3.6).* 
*Report any clinically important increase in the rate of a serious suspected 
adverse reaction (at your study site) over that which is listed in the 
protocol or investigator brochure as an expedited event.  
*Report an expected event that is greater in severity or specificity than 
expected as an expedited event. 
*Specific protocol exceptions to expedited reporting should be reported 
expeditiously by investigators ONLY if they exceed the expected grade of 
the event. 
CTCAE 
System Organ Class 
(SOC) 
Adverse event/ 
Symptoms 
CTCAE Grade at which the 
event will not be reported in an 
expedited manner1 
General disorders and 
administrations site 
conditions 
Fatigue  
≤Grade 3 
Malaise 
≤Grade 3 
Skin and subcutaneous 
tissue disorders 
Alopecia 
≤Grade 4 
1 These exceptions only apply if the adverse event does not result in hospitalization. If the 
adverse event results in hospitalization, then the standard expedited adverse events 
reporting requirements must be followed.  

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The following hospitalizations are not considered to be SAEs because there is no 
“adverse event” (i.e., there is no untoward medical occurrence) associated with 
the hospitalization: 
• Hospitalizations for respite care 
• Planned hospitalizations required by the protocol 
• Hospitalization planned before informed consent (where the condition 
requiring the hospitalization has not changed post study drug 
administration) 
• Hospitalization for elective procedures unrelated to the current disease 
and/or treatment on this trial 
• Hospitalization for administration of study drug or insertion of access for 
administration of study drug 
• Hospitalization for routine maintenance of a device (e.g., battery 
replacement) that was in place before study entry 
• ● Hospitalization, or other serious outcomes for signs and symptoms of 
progression of the cancer. 
 Serious Adverse Events 
All serious adverse events, regardless of causality to study drug, will be reported to the 
Principal Investigator and the Study Coordinator at each institution, and also to the 
Coordinating Center. 
All serious adverse events must be reported to the Coordinating Center within 1 business 
day after the investigator becomes aware of the event. Events should be reported using 
a MedWatch form (3500A) as available on the FDA website (see link below).  
Follow-up information must also be reported within 1 business day of receipt of the 
information by the investigator. 
The Coordinating Center will disseminate information regarding serious adverse events 
to the participating sites within 5 days of review of the information by the Protocol 
Chair (or her designee in the event of extended absence) only in the case that the 
event(s) is believed to be related (i.e., possibly, probably, or definitely) to the study 
medication. The Coordinating Center will be responsible for reporting of events to the 
FDA and supporters, as appropriate (outlined below). 
 General reporting instructions 
The Mayo IND and/or MCCC Compliance will assist the sponsor-investigator in 
the processing of expedited adverse events and forwarding of suspected 
unexpected serious adverse reactions (SUSARs) to the FDA and IRB. 
13.3.3.1.1 Mayo Clinic Sites 
Mayo sites will use Mayo Expedited Event Report form 
http://livecycle2.mayo.edu/workspace/?startEndpoint=MC4158-
56/Processes/MC4158-56-Process.MC4158-56 for investigational agents or 
commercial/investigational agents on the same arm.  
Attach the MedWatch 3500A form to the Mayo Expedited Event Report form 
http://livecycle2.mayo.edu/workspace/?startEndpoint=MC4158-
56/Processes/MC4158-56-Process.MC4158-56.  

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 Expedited Reporting Requirements for IND Agents  
 Late Phase 2 and Phase 3 Studies: Expedited Reporting Requirements 
for Adverse Events that Occur on Studies under an IND within 30 
Days of the Last Administration of the Investigational 
Agent/Intervention1, 2 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE: Investigators MUST immediately report to the sponsor ANY Serious Adverse Events, whether or not they 
are considered related to the investigational agent(s)/intervention (21 CFR 312.64) 
 An adverse event is considered serious if it results in ANY of the following outcomes:  
1) 
Death 
2) 
A life-threatening adverse event  
3) 
An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 
hours  
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  
5) 
A congenital anomaly/birth defect.  
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon medical judgment, they may jeopardize the patient or 
subject and may require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the sponsor within 
the timeframes detailed in the table below. 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
Hospitalization 
≥24 hrs 
7 Calendar Days 
24-Hour  
3 Calendar 
Days 
Not resulting in 
Hospitalization 
≥24 hrs 
Not required 
7 Calendar Days 
Expedited AE reporting timelines are defined as: 
o “24-Hour; 3 Calendar Days” - The AE must initially be reported within 24 hours of learning of the AE, 
followed by a complete expedited report within 3 calendar days of the initial 24-hour report. 
o “7 Calendar Days” - A complete expedited report on the AE must be submitted within 7 calendar days 
of learning of the AE. 
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention and have an attribution of possible, probable, or definite require reporting as follows:  
Expedited 24-hour notification followed by complete report within 3 calendar days for: 
• All Grade 4, and Grade 5 AEs 
Expedited 7 calendar day reports for: 
• Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
• Grade 3 adverse events 
2 For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-lives, 
rounded UP to the nearest whole day, after the agent/intervention was last administered. Footnote “1” above 
applies after this reporting period. 
Effective Date: May 5, 2011 
NOTE: Refer to Section 13.3.2 for Exceptions to Expedited Reporting 

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 Other Required Reporting 
 Unanticipated Problems Involving Risks to Subjects or Others 
(UPIRTSOS) 
Unanticipated Problems Involving Risks to Subjects or Others (UPIRTSOS) in 
general, include any incident, experience, or outcome that meets all of the following 
criteria: 
1. Unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
2. Related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
3. Suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was 
previously known or recognized. 
Some unanticipated problems involve social or economic harm instead of the 
physical or psychological harm associated with adverse events. In other cases, 
unanticipated problems place subjects or others at increased risk of harm, but no 
harm occurs. 
Note: If there is no language in the protocol indicating that pregnancy is not 
considered an adverse experience for this trial, and if the consent form does not 
indicate that subjects should not get pregnant/impregnate others, then any 
pregnancy in a subject/patient or a male patient’s partner (spontaneously reported) 
which occurs during the study or within 120 days of completing the study should be 
reported as a UPIRTSO. 
Mayo Clinic Cancer Center (MCCC) Institutions: 
If the event meets the criteria for IRB submission as a Reportable Event/UPIRTSO, 
provide the appropriate documentation and use the Mayo Clinic Cancer Center 
Expedited Event Report form 
http://livecycle2.mayo.edu/workspace/?startEndpoint=MC4158-
56/Processes/MC4158-56-Process.MC4158-56, to submit to 
CANCERCROSAFETYIN@mayo.edu. The Mayo Clinic Compliance Unit will 
review and process the submission to the Mayo Clinic IRB and work with the IND 
Coordinator for submission to FDA. 
 Death 
Note: A death on study requires both routine and expedited reporting 
regardless of causality, unless as noted below. Attribution to treatment or 
other cause must be provided. 
Any death occurring within 30 days of the last dose, regardless of attribution to 
an agent/intervention under an IND requires expedited reporting within 24-
hours. 

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Any death occurring greater than 30 days with an attribution of possible, 
probable, or definite to an agent/intervention under an IND requires expedited 
reporting within 24-hours. 
Reportable categories of Death  
• Death attributable to a CTCAE term. 
• Death Neonatal: A disorder characterized by cessation of life during the 
first 28 days of life.  
• Death not otherwise specified (NOS): A cessation of life that cannot be 
attributed to a CTCAE term associated with Grade 5. 
• Sudden death NOS: A sudden (defined as instant or within one hour of the 
onset of symptoms) or an unobserved cessation of life that cannot be 
attributed to a CTCAE term associated with Grade 5. 
• Death due to progressive disease should be reported as Grade 5 
“Neoplasms benign, malignant and unspecified (including cysts and 
polyps) – Other (Progressive Disease)” under the system organ class 
(SOC) of the same name. Evidence that the death was a manifestation of 
underlying disease (e.g., radiological changes suggesting tumor growth or 
progression: clinical deterioration associated with a disease process) should 
be submitted. 
 Secondary Malignancy 
• A secondary malignancy is a cancer caused by treatment for a previous 
malignancy (e.g., treatment with investigational agent/intervention, radiation 
or chemotherapy). A secondary malignancy is not considered a metastasis of 
the initial neoplasm. 
• All secondary malignancies that occur following treatment with an agent under 
an IND will be reported. Three options are available to describe the event: 
o Leukemia secondary to oncology chemotherapy (e.g., Acute Myelocytic 
Leukemia [AML]) 
o Myelodysplastic syndrome (MDS) 
o Treatment-related secondary malignancy 
• Any malignancy possibly related to cancer treatment (including AML/MDS) 
should also be reported via the routine reporting mechanisms outlined in each 
protocol. 
 Second Malignancy 
A second malignancy is one unrelated to the treatment of a prior malignancy (and is 
NOT a metastasis from the initial malignancy). Second malignancies require ONLY 
routine reporting unless otherwise specified. 
 Pregnancy, Fetal Death, and Death Neonatal  
If a female subject (or female partner of a male subject) taking investigational 
product becomes pregnant, the subject taking should notify the Investigator, and the 
pregnant female should be advised to call her healthcare provider immediately. The 

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patient should have appropriate follow-up as deemed necessary by her physician. If 
the baby is born with a birth defect or anomaly, a second expedited report is 
required. 
Prior to obtaining private information about a pregnant woman and her infant, the 
investigator must obtain consent from the pregnant woman and the newborn 
infant’s parent or legal guardian before any data collection can occur. A consent 
form will need to be submitted to the IRB for these subjects if a pregnancy occurs. 
If informed consent is not obtained, no information may be collected. 
In cases of fetal death, miscarriage or abortion, the mother is the patient. In cases 
where the child/fetus experiences a serious adverse event other than fetal death, the 
child/fetus is the patient. 
NOTE: When submitting Mayo Expedited Adverse Event Report reports for 
“Pregnancy”, “Pregnancy loss”, or “Neonatal loss”, the potential risk of exposure of 
the fetus to the investigational agent(s) or chemotherapy agent(s) should be 
documented in the “Description of Event” section. Include any available medical 
documentation. Include this form: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/Pregnanc
yReportFormUpdated.pdf 
13.3.5.5.1 Pregnancy 
Pregnancy should be reported in an expedited manner as Grade 3 
“Pregnancy, puerperium and perinatal conditions - Other 
(pregnancy)” under the Pregnancy, puerperium and perinatal conditions 
SOC. Pregnancy should be followed until the outcome is known. 
13.3.5.5.2 Fetal Death 
Fetal death is defined in CTCAE as “A disorder characterized by death in 
utero; failure of the product of conception to show evidence of respiration, 
heartbeat, or definite movement of a voluntary muscle after expulsion 
from the uterus, without possibility of resuscitation.” 
Any fetal death should be reported expeditiously, as Grade 4 
“Pregnancy, puerperium and perinatal conditions - Other (pregnancy 
loss)” under the Pregnancy, puerperium and perinatal conditions SOC. 
13.3.5.5.3 Death Neonatal 
Neonatal death, defined in CTCAE as “A disorder characterized by 
cessation of life occurring during the first 28 days of life” that is felt by 
the investigator to be at least possibly due to the investigational 
agent/intervention, should be reported expeditiously.  
A neonatal death should be reported expeditiously as Grade 4 “General 
disorders and administration - Other (neonatal loss)” under the 
General disorders and administration SOC.  

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 Required Routine Reporting - Cohort B Group 4 ONLY 
 Baseline and Follow-up Adverse Events Evaluations  
Pretreatment symptoms/conditions to be graded at baseline and adverse events to 
be graded at each evaluation.  
Grading is per CTCAE v5.0. 
CTCAE  
System/Organ/Class 
(SOC) 
Adverse event/Symptoms 
Baseline 
Prior to 
Surgery 
End of 
Study 
Blood and lymphatic 
system disorders 
Anemia 
X 
X 
 
Gastrointestinal disorders 
Baseline # of stools 
X 
 
 
Diarrhea 
 
X 
X 
Nausea 
X 
X 
X 
Infections and infestations Wound infection 
 
 
X 
Injury, poisoning and 
Seroma 
 
 
X 
procedural complications 
Wound complication 
 
 
X 
Investigations 
Alanine aminotransferase increased 
X 
X 
 
Blood bilirubin increased 
X 
X 
 
Aspartate aminotransferase increased 
X 
X 
 
 
Neutrophil count decreased 
X 
X 
 
 
Platelet count decreased 
X 
X 
 
Vascular disorders 
Hematoma 
 
 
X 
Thromboembolic event 
 
X 
X 
 All other AEs 
Submit via appropriate MCCC Case Report Forms (i.e., paper or electronic, as 
applicable) the following AEs experienced by a patient and not specified in 
Section 13.3.6.1: 
13.3.6.2.1 Grade 2 AEs deemed possibly, probably, or definitely related to the study 
treatment or procedure. 
13.3.6.2.2 Grade 3 and 4 AEs regardless of attribution to the study treatment or 
procedure. 
13.3.6.2.3 Grade 5 AEs (Deaths) 
13.3.6.2.3.1 Any death within 30 days of the patient’s last study treatment or 
procedure regardless of attribution to the study treatment or 
procedure. 
13.3.6.2.3.2 Any death more than 30 days after the patient’s last study 
treatment or procedure that is felt to be at least possibly treatment 
related must also be submitted as a Grade 5 AE, with a CTCAE 
type and attribution assigned. 

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 Late Occurring Adverse Events 
Refer to the instructions in the Forms Packet (or electronic data entry screens, as 
applicable) regarding the submission of late occurring AEs following completion of 
the Active Monitoring Phase (i.e., compliance with Test Schedule in Section 4.0). 
 Institutional Review Board 
All adverse events and serious adverse events will be reported to the Mayo Clinic IRB 
per current institutional standards. If an adverse event requires modification of the 
informed consent, these modifications will be provided to the IRB with the report of 
the adverse event. If an adverse event requires modification to the study protocol, these 
modifications will be provided to Mayo Clinic as soon as possible. 
 Food and Drug Administration (FDA) 
Mayo Clinic Cancer Center Compliance has been designated to manage the 
Investigational New Drug Application (IND) associated with this protocol on behalf of 
the sponsor-investigator. Mayo Clinic Cancer Center Compliance will cross-reference 
this submission to Eli Lilly and Company’s parent IND at the time of submission. 
Additionally, Mayo Clinic Cancer Center Compliance will submit a copy of these 
documents to Eli Lilly and Company at the time of submission to FDA. 
Sponsor-Investigator will be responsible for all communication with the FDA in 
accordance with 21CFR312, which includes but is not limited to the 7 and 15 Day 
Reports, as well as an Annual Progress Report. Additionally, Mayo Clinic Cancer 
Center Compliance will submit a copy of these reports to Eli Lilly and Company at the 
time of submission to FDA. 
In this trial, unexpected adverse events believed to be definitely, probably, or possibly 
related to the medications will be reported to the Food and Drug Administration via 
MedWatch. The Coordinating Center will be responsible for correspondence 
regarding adverse events with the FDA for all participating sites. Sites will be 
instructed in the method by which to report events to the Coordinating Center and per 
what forms (i.e., mandatory MedWatch 3500A forms available at: 
http://www.fda.gov/downloads/AboutFDA/ReportsManualsForms/Forms/UCM048334.pdf 
 Other 
All sites will report SAEs as stipulated by Eli Lilly and Company (i.e., all SAEs, only 
those related, etc.) within 1 business day of receipt of the SAE Reporting Form. 
Follow-up information will be provided to Eli Lilly and Company as reasonably 
requested. 
14. DATA MONITORING 
14.1 Monitoring 
A data and safety monitoring plan (DSMP) will be conducted under the auspices of the 
lead/primary institution, Mayo Clinic. 
Data monitoring including on-site audits will be conducted under the auspices of the 
lead/primary institution, Mayo Clinic, and its assignees. 

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15. REGULATORY CONSIDERATIONS 
15.1 Protocol Review and Amendments 
Information regarding study conduct and progress will be reported to the Mayo Clinic 
Institutional Review Board (IRB) per the current institutional standards. 
Any changes to the protocol will be made in the form of an amendment and must be 
approved by the Mayo Clinic IRB prior to implementation.  
The Protocol Chairs (or their designee) is responsible for the coordination and development 
of all protocol amendments, and will disseminate this information to the participating 
centers. 
15.2 Informed Consent 
The investigator (or his/her designee) will explain to each subject the nature of the study, 
its purpose, the procedures involved, the expected duration, the potential risks and benefits 
involved and any discomfort it may entail. Each subject will be informed that participation 
in the study is voluntary, that s/he may withdraw from the study at any time, and that 
withdrawal of consent will not affect her subsequent medical treatment or relationship with 
the treating physician(s) or institution. The informed consent will be given by means of a 
standard written statement, written in non-technical language, which will be IRB approved. 
The subject should read and consider the statement before signing and dating it, and will 
be given a copy of the document. No subject will enter the study or have study-specific 
procedures done before his/her informed consent has been obtained. 
In accordance with the Health Information Portability and Accountability Act (HIPAA), 
the written informed consent document (or a separate document to be given in conjunction 
with the consent document) will include a subject authorization to release medical 
information to the study sponsor and supporting agencies and/or allow these bodies, a 
regulatory authority, or Institutional Review Board access to subjects’ medical information 
that includes all hospital records relevant to the study, including subjects’ medical history. 
15.3 Ethics and GCP 
This study will be carried out in compliance with the protocol and Good Clinical Practice, 
as described in: 
1. ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
2. US 21 Code of Federal Regulations dealing with clinical studies (including parts 50 
and 56 concerning informed consent and IRB regulations). 
3. Declaration of Helsinki, concerning medical research in humans (Recommendations 
Guiding Physicians in Biomedical Research Involving Human Subjects, Helsinki 1964, 
amended Tokyo 1975, Venice 1983, Hong Kong 1989, Somerset West 1996). 
The investigator agrees to adhere to the instructions and procedures described in it and 
thereby to adhere to the principles of Good Clinical Practice that it conforms to. 

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15.4 Compliance with Trial Registration and Results Posting Requirements 
Under the terms of the Food and Drug Administration Modernization Act (FDAMA) and 
the Food and Drug Administration Amendments Act (FDAAA), the sponsor-investigator 
of the trial is solely responsible for determining whether the trial and its results are subject 
to 
the 
requirements 
for 
submission 
to 
the 
Clinical 
Trials 
Data 
Bank, 
http://www.clinicaltrials.gov. Information posted will allow subjects to identify potentially 
appropriate trials for their disease conditions and pursue participation by calling a central 
contact number for further information on appropriate trial locations and study site contact 
information. 
16. STUDY CONDUCT GUIDELINES 
16.1 Study Documentation 
Each participating site is responsible for submitting copies of all relevant regulatory 
documentation. The required documents include, but are not limited to the following:local 
Delegation of Authority (DOA), financial disclosures, summary of unanticipated problems 
or protocol deviations, and documentation of expertise of the investigators. It is the 
responsibility of the participating sites to maintain copies of all documentation submitted. 
16.2 Records Retention 
Following closure of the study, each participating center will maintain a copy of all site 
study records in a safe and secure location. 
16.3 Publication 
It is understood that any manuscript or releases resulting from the collaborative research 
must be approved by the Protocol Chair and will be circulated to applicable participating 
sites/investigators prior to submission for publication or presentation.  
Additionally, any publication of study data and results must conform to the Mayo Clinic 
Cancer Center publications policy. 
 

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17. STATISTICAL CONSIDERATIONS 
17.1 Background 
Miyashita et al demonstrated among women with triple negative breast cancer who 
received neo-adjuvant chemotherapy that recurrence-free survival was significant longer 
for those women with a CD8/FOXP3 ratio ≥1.6 in their residual tumors than those 
women with a CD8/FOXP3 ratio <1.6 in their residual tumors (HRadj =2.07; 95%CI: 
1.03-4.44). When CD8/FOXP3 ratio was considered on a continuous scale after adjusting 
for other prognositic factors, it was not found to be significantly associated with RFS 
(reference: Breast Cancer Res 2015; 16(1):124) 
Karihtala et al found that approximately 64% of primary triple negative breast cancers 
express vimentin and vimentin expression is associated with both high proliferation rates 
and high tumor grade. Unpublished Mayo Clinic data evaluating staining of vimentin in a 
cohort of 30 TNBC patients treated with AC followed by paclitaxel who had residual 
disease demonstrated a similar frequency of vimentin expression (around 60%). 
Data from the NeoPalAna study (Ma et al) of women receiving neoadjuvant palbociclib 
with anastrozole for ER+ breast cancer reported that those women who did not have 
surgery the day after their last dose of palbociclib (median 29 days from the last dose of 
palbociclib) exhibited significantly higher Ki-67 at surgery compared to patients who 
went to surgery immediately after the last dose of palbociclib.49 
As such, assessment of the primary endpoint will include all eligible women with a pre-
abemaciclib CD8/FOXP3 ratio < 1.6 who completed 14-21 days of abemaciclib and 
underwent surgery or breast biopsy at most 3 days after last dose of abemaciclib (referred 
to as evaluable patients). 
17.2 Study Design 
The primary aim of this clinical trial is to examine the effect of abemaciclib on the 
CD8/FOXP3 ratio in two patient populations, namely, patients with chemotherapy 
resistant TNBC following neoadjuvant chemotherapy (Cohort A) and patients with 
patients with chemotherapy resistant TNBC following neoadjuvant chemotherapy with 
pembrolizumab (Cohort B).  
Specifically, for each cohort, we seek to estimate the proportion of patients with a pre-
abemaciclib CD8/FOXP3 ratio < 1.6 who completed 14-21 days of abemaciclib and 
underwent surgery or breast biopsy at most 3 days after last dose of abemaciclib who 
have a post-abemaciclib CD8/FOXP3 ratio ≥1.6 in their residual tumors  
For each cohort, a Simon optimum two stage phase II clinical trial design was chosen to 
test the null hypothesis that the rate of conversion to post-abemaciclib CD8/FOXP3 ratio 
of 1.6 or more is ≤5% against the alternative that rate of conversion to post-abemaciclib 
CD8/FOXP3 ratio of 1.6 or more is ≥20% with the target probability of a type I error and 
probability of a type II error set to 0.10. The probability of terminating after the first stage 
is 0.736, if the null hypothesis is true. 

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Stage 1: Twenty evaluable patients will be enrolled and then registration will be 
temporarily halted until there are sufficient post-abemaciclib CD8/FOXP3 ratio 
results to evaluate the decision rule.  
• If at most 1 of these 20 patients is found to have a post-abemaciclib 
CD8/FOXP3 ratio ≥1.6, then enrollment will be closed and a short course of 
abemaciclib following NAC will not be considered promising in increasing 
post-NAC CD8/FOXP3 ratios above the threshold of 1.6 in this patient 
population.  
• If at 2 or more of these 20 patients is found to have a post-abemaciclib 
CD8/FOXP3 ratio ≥1.6, then enrollment will reopen to obtain additional 15 
patients. 
Stage 2: Fifteen patients will be enrolled with a post-NAC CD8/FOXP3 ratio 
<1.6.  
• If at most 3 of these 35 patients are found to have a post-abemaciclib 
CD8/FOXP3 ratio ≥1.6, then a short course of abemaciclib following 
NAC will not be considered promising in increasing post-NAC 
CD8/FOXP3 ratios above the threshold of 1.6 in this patient population.  
• If 4 or more of these 35 patients are found to have a post-abemaciclib 
CD8/FOXP3 ratio ≥1.6, then this approach will be recommended for 
further testing. 
Note: Post-NAC CD8/FOXP3 ratios will not be known at the time of registration. 
Miyashita et al reported that at 50% of patients with TNBC will have a post-NAC 
CD8/FOXP3 ratio <1.6. As such we will begin by registering as many as 40 patients onto 
the first stage of the trial to obtain 20 patients with a post-NAC CD8/FOXP3 ratio <1.6. 
This assumption will be monitored throughout the first stage of the enrollment period. A 
protocol amendment will be drafted for DSMB and IRB approval if this assumption was 
too pessimistic or optimistic. If the assumption that 50% of the patients enrolled will have 
a post-NAC CD8/FOXP3 ratio <1.6 and there is sufficient evidence of activity from the 
first stage to open the trial to the second stage, 30 patients will be enrolled to obtain 15 
patients with a post-NAC CD8/FOXP3 ratio <1.6. 
A 90% confidence interval for this rate of conversion will be constructed using the 
Duffy-Santner approach taking into account the sequential nature of the study design. 
As of Amendment 6, enrollment to Cohort A was closed related to a change in the 
clinical practice with neoadjuvant pembrolizumab now standardly utilized in combination 
with chemotherapy. At the time of closure to Cohort A, 10 patients had been enrolled. As 
such, a 90% binomial confidence for the rate of conversion among the Cohort A patients 
will be constructed. 
17.3 Secondary Endpoints and analysis plans for each cohort independently 
 Adverse events 
All patients who begin abemaciclib treatment will be included in the summary of 
adverse events. 

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Prior to and at discontinuation of abemaciclib treatment, adverse events will be 
monitored and graded with attribution assigned using CTCAE version 5.0. For each 
type of adverse event (AE) reported, the proportion of patients experiencing a Grade 
2 or worse level of that AE will be determined.  
 Changes in Vimentin expression 
Based on findings of Karihtala et al. and the Mayo team, approximately 60% of the 
patients enrolled are expected to have post NAC vimentin expressing invasive cancer 
cells. Vimentin expression will be evaluated by IHC with using 0, 1+, 2+ or 3+ 
scoring scheme. 
A point estimate of the proportion of eligible patients whose post-NAC residual 
tumor Vimentin expression levels were 2+ or 3+ and then fell to 0 or 1+ after 
abemaciclib among the eligible patients whose began abemaciclib treatment after a 
post NAC residual tumor Vimentin expression level finding of 2+ or 3+ will be 
calculated. A 90% confidence interval for this proportion will be constructed using 
one-sample binomial confidence for proportions. 
 Exploration of impact of length of treatment 
Association between amount of abemaciclib received and the change in CD8/FOXP3 
ratio after abemaciclib will be explored graphically. A plot of the change in change in 
CD8/FOXP3 ratio after abemaciclib and days of treatment will be constructed to 
visually assess for trends. 
Also, the a 90% confidence interval for the difference in binomial proportions will be 
constructed to assess whether the proportion of patients whose post abemaciclib 
CD8/FOXP3 ratio ≥1.6 after completing 14-21 days of abemaciclib differs between 
those who discontinued abemaciclib the day prior to surgery and who discontinued 
abemaciclib 2 or more days prior to surgery 
A 90% binomial confidence interval for the proportion of women who failed to 
complete 14-21 days of abemaciclib or underwent surgery or breast biopsy 2 or more 
days after last dose of abemaciclib among the eligible women who began abemaciclib 
treatment. 
17.4 Correlative Endpoints for each cohort independently 
Changes in mitoses, nuclear pleomorphism, and tubule formation, tumor Ki-67, serum 
TKI, SNAIL, SLUG, E-Cadherin, and tumor-infiltrating lymphocytes may be 
examined graphically overall and by TNBC subtypes. 
17.5 Exploratory objectives for each cohort independently 
Effect of abemaciclib on tumor RNA sequencing followed by standard and 
customized pathway and enrichment analysis (e.g., Gene set enrichment analysis 
(GSEA) and CIBERSORT methods). The GSEA method determines whether an a 
priori set of genes are over-represented in the differential expression analysis of the 
pre- and post- drug response phenotypes. Similarly, CIBERSORT estimates cell 
composition from RNA transcriptomic data (e.g. immune cell populations). 

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The effects of abemaciclib on FFPE tumor sections (imaging mass cytometry multiplex 
quantitative RNA/protein immune-based profilings, such as Hyperion TM, Nanostring 
GeoMX, Codex (all available at Mayo Clinic)) which may include examination of: 
• 
genes directly involved in tumor cell antigen presentation (e.g. B2M, HLA-A, 
HLA-B, HLA-C, TAP1, TAP2, TAPBP) 
• 
interferon-stimulated genes (ISGs) that regulate antigen presentation (e.g. 
STAT1, NLRC5) and other ISGs (e.g. IRFs, OAS2) 
• 
genes involved in dsRNA response (e.g. DDX58, DHX58) 
• 
genes encoding interferons, including type 3 IFNs (e.g. IFNL1, IFNL2, IFNL3) 
• 
genes indicating a cytotoxic T cell response (e.g. PRF1, GZMB) 
• 
Treg-specific transcription factor genes (e.g., FOXP3, IKZF2). 
The effects of abemaciclib on the peripheral blood immune phenotype will be assessed 
using mass cytometry (e.g,. CyTOF) to analyze a panel of 29 cell surface markers 
optimized for identification of human immune cell subsets, including the following 
targets: CD45, CD195 (CCR6), CD19, CD127, CD38, IgD, CD11c, CD16, CD194 
(CCR4), CD123/IL-3R, TCRgd, CD185 (CXCR5), CD3, CD45RA, CD27, CD29, 
CD66b, CD183 (CXCR3), CD161, CD45RO, CD197 (CCR7), CD8a, CD25/IL-2R, 
CD20, HLA-DR, CD4, CD14, CD56/NCAM. Identification of specific immune cell 
subsets will be assessed using consensus clustering methods of pre-defined 
combination of markers.  
The collection of JAK-2 will be used to assess the difference in the frequency of JAK-
2 amplification among patients whose post-abemaciclib CD8/FOXP3 ratio ≥1.6 and 
that among patients whose post-abemaciclib CD8/FOXP3 ratio <1.6. A 95% binomial 
confidence for the difference in two independent proportions will be constructed for 
the difference in the percentage of patients with a post-abemaciclib CD8/FOXP3 ratio 
≥1.6 among those with JAK-2 amplified TNBC and those with JAK-2 non-amplified 
TNBC. 
The collection of microbiome information via stool collection will be used to generate 
descriptive analyses about the study population, as the total number of samples is 
expected to be insufficient for full statistical analysis. We will explore putative 
mechanistic connections underlying bacteria-drug interactions in all patients and 
attempt to identify the biomolecular features within the gut (stool) microbiome and its 
association with the pharmacokinetics and pharmacodynamics of abemaciclib. 
17.6 Accrual and study duration  
With the closure of Cohort A, we plan to continue to pre-register 104 TNBC patients 
who completed an NAC regimen that included pembrolizumab. Those patients with 
evidence of residual disease on imaging are eligible to register onto the study. wWe 
anticipate that 25 patients will be found to have no invasive disease in their biopsy 
specimen and will go directly to surgery. 
Of the remaining 78 patients with residual invasive disease who register to the trial and 
begin abemaciclib, we anticipate that 50% of these patients will have a pre-abemaciclib 
a CD8/FOXP3 ratio ≥1.6 and another 5% will fail to complete 14 to 21 days of 
abemaciclib within 3 days of surgery, leaving 35 evaluable patients.  

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We anticipate that the rate of patient registration will be 2-3 patients per month. Accrual 
period will be approximately 30 months. The analysis of the primary endpoint is 
expected to be completed approximately 6 months thereafter. 
17.7 Safety monitoring 
The study chair and study statistician will review the trial data every 3 months to 
identify accrual, adverse events, and feasibility isuse that might be developing. The 
study statistician will prepare a report containing accrual, adverse events (including 
whether the safety threshold has been crossed) and study conduct issues which will be 
submitted to the MCCC Data and Safety Monitoring Board on a semi-annual basis. 
The safety threshold will be examined utilizing all eligible patients who started protocol 
treatment. 
 Safety Threshold for each cohort 
If 3 or more of the first 10 patients enrolled or 30% of more of patients enrolled 
thereafter develops a Grade 4 adverse event (AE) possibly, probably or 
definitely related to treatment, the enrollment to the trial will be temporarily 
suspended so that all AE data can be examined. The study chair and the study 
statistician will formulate a trial recommendation to present to the MCCC 
DSMB for approval. 
17.8 Inclusion of Women and Minorities 
This study will be available to all eligible patients regardless of race or ethnic group. 
The expected number of patients per racial/ethnicity categories are presented in the 
following table. The sample size for this trial was not increased in order to provide 
additional power for analyses by race or ethnicity. 
NOTE: We have excluded men from enrollment in this trial for two reasons:  
1) Breast cancer is not common in males so only one or two patients are 
likely to be enrolled; and  
2) Because this trial is small, the addition of one or two male patients may 
confound the results without providing meaningful data. 
Accrual Targets for Cohort B ONLY 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
11 
 
 
Not Hispanic or Latino 
93 
 
 
Ethnic Category: Total of all subjects 
104 
0 
104 
Racial Category 
American Indian or Alaskan Native 
0 
 
 
Asian 
3 
 
 
Black or African American 
3 
 
 
Native Hawaiian or other Pacific Islander 
0 
 
 
White 
98 
 
 
Racial Category: Total of all subjects 
104 
0 
104 
 

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Ethnic 
Categories: 
Hispanic or Latino – a person of Cuban, Mexican, Puerto Rican, South or Central 
American, or other Spanish culture or origin, regardless of race. The term “Spanish 
origin” can also be used in addition to “Hispanic or Latino.” 
Not Hispanic or Latino 
Racial 
Categories: 
American Indian or Alaskan Native – a person having origins in any of the original 
peoples of North, Central, or South America, and who maintains tribal affiliations or 
community attachment. 
Asian – a person having origins in any of the original peoples of the Far East, 
Southeast Asia, or the Indian subcontinent including, for example, Cambodia, China, 
India, Japan, Korea, Malaysia, Pakistan, the Philippine Islands, Thailand, and 
Vietnam. (Note: Individuals from the Philippine Islands have been recorded as Pacific 
Islanders in previous data collection strategies.)  
Black or African American – a person having origins in any of the black racial 
groups of Africa.  
Native Hawaiian or other Pacific Islander – a person having origins in any of the 
original peoples of Hawaii, Guam, Samoa, or other Pacific Islands. 
White – a person having origins in any of the original peoples of Europe, the Middle 
East, or North Africa. 
18. BUDGET 
18.1 Costs charged to patient 
Routine clinical care 
18.2 Treatment and tests to be research funded 
Abemaciclib, research blood work, and tumor biopsy at pre-registration (and if 
surgery is not done) 
 
 
 

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platform integrated with automated immunofluorescence staining. BMC Cancer 
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19.1 Additional References for Translational Studies 
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Gopalakrishnan V, S. C. (2018). Gut microbiome modulates response to anti-PD-1 
immunotherapy in melanoma patients. Science, 359 (6371):97-103. 
Kwa M, P. C. (2016). The Intestinal Microbiome and Estrogen Receptor-Positive Female Breast 
Cancer. Journal of the National Cancer Institute, 108(8). 
Routy B, L. C. (2018). Gut microbiome influences efficacy of PD-1-based immunotherapy 
against epithelial tumors. Science, 359(6371)91-7. 
 
 
 

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Protocol Chair: Matthew P Goetz MD/Judy C Boughey MD 
MC1734 
 
 
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APPENDICES  
Appendix A: ECOG Performance Status Scale 
Appendix B: Study Drug Diary 
Appendix C: Lilly Guidance for Hepatic Treatment Emergent Abnormality 
 
 

Window trial Abemaciclib TNBC 
 
MCCC Amendment 7 
Protocol Chair: Matthew P Goetz MD/Judy C Boughey MD 
MC1734 
 
 
Protocol Version: 20Feb2024 
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APPENDIX A: ECOG-Karnofsky Performance Status Scale 
Grade 
ECOG Definition* 
Karnofsky 
Definition 
Karnofsky 
Equivalent 
0 
Fully active, able to carry on all pre-disease 
performance without restriction 
Asymptomatic 
100 
1 
Restricted in physically strenuous activity but 
ambulatory and able to carry out work of a light or 
sedentary nature, e.g., light house work, office 
work 
Symptomatic, fully 
ambulatory 
80 – 90 
2 
Ambulatory and capable of all selfcare but unable 
to carry out any work activities. Up and about 
more than 50% of waking hours 
Symptomatic, in bed 
less than 50% of day 
60 – 70 
3 
Capable of only limited selfcare, confined to bed 
or chair more than 50% of waking hours. 
Symptomatic, in bed 
more than 50% of 
day,  
but not bedridden 
40 – 50 
4 
Completely disabled. Cannot carry on any 
selfcare. Totally confined to bed or chair. 
Bedridden 
20 – 30 
5 
Dead 
 
 
*As published in Am. J. Clin. Oncol.: 
Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., McFadden, E.T., Carbone, P.P.: Toxicity And 
Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol 5:649-655, 1982. 
The ECOG Performance Status is in the public domain therefore available for public use. To duplicate the scale, 
please cite the reference above and credit the Eastern Cooperative Oncology Group, Robert Comis M.D., Group 
Chair. 
From http://www.ecog.org/general/perf_stat.html 
 
 
 

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APPENDIX B: Study Drug Diary (Group 4 only) 
Document removed - The study drug diary is provided as a standalone document as required by 
the Mayo Clinic Foundation Institutional Review Board (IRB). This version is removed to avoid 
the appearance of conflicting versions in the case of audit. 

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APPENDIX C: Lilly Guidance for Hepatic Treatment Emergent Abnormality 
Liver testing including ALT, AST, alkaline phosphatase (ALP), total bilirubin (TBL), direct 
bilirubin (D. Bil), gamma-glutamyltransferase (GGT), and creatine kinase (CK), should be 
repeated within 2 to 4 days to confirm the abnormality and to determine if it is increasing or 
decreasing, if one or more of these conditions occur: 
If a participant with baseline 
results of...  
develops the following elevations: 
ALT or AST <1.5×ULN 
ALT or AST ≥5×ULN or 
ALT or AST ≥3×ULN concurrent with TBL ≥2×ULN 
 
  
ALT or AST ≥1.5×ULN 
ALT or AST ≥3× baseline or 
ALT or AST ≥2× baseline concurrent with TBL 
≥2×ULN 
If the abnormality persists or worsens, clinical and laboratory monitoring and evaluation for 
possible causes of abnormal liver tests, should be initiated by the investigator. At a minimum, 
this evaluation should include physical examination and a thorough medical history, including 
symptoms, recent illnesses (for example, heart failure, systemic infection, hypotension, or 
seizures), history of concomitant medications (including over-the-counter, herbal and dietary 
supplements, history of alcohol drinking and other substance abuse). In addition, the evaluation 
should include a blood test for prothrombin time (PT-INR); serological tests for viral hepatitis A, 
B, C, E, autoimmune hepatitis; and an abdominal imaging study (for example, ultrasound or CT 
scan). 
Based on the patient’s history and initial evaluation results, further testing should be considered, 
including tests for hepatitis D virus (HDV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), 
acetaminophen levels, acetaminophen protein adducts, urine toxicology screen, Wilson’s disease, 
blood alcohol levels, urinary ethyl glucuronide, and blood phosphatidylethanol. Based on the 
circumstances and the investigator’s assessment of the participant’s clinical condition, the 
investigator should consider referring the participant for a hepatologist or gastroenterologist 
consultation, magnetic resonance cholangiopancreatography (MRCP), endoscopic retrograde 
cholangiopancreatography (ERCP), cardiac echocardiogram, and/or a liver biopsy. 
Additional Hepatic Safety Data Collection 
Additional safety data (Table 8.4.3) should be collected via the CRF if 1 or more of the 
following conditions occur:  
In participants enrolled with baseline ALT or AST <1.5×ULN 
• Elevation of serum ALT or AST to ≥5×ULN on 2 or more consecutive blood tests 
• The combination of elevated ALT or AST ≥3×ULN and elevated TBL ≥2×ULN 
In participants enrolled with baseline ALT or AST ≥1.5×ULN 
• Elevated ALT or AST ≥3× baseline on 2 or more consecutive tests 
• The combination of elevated ALT or AST ≥2× baseline and elevated TBL ≥2×ULN 

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In all study participants  
• Discontinuation from study intervention due to a hepatic event or abnormality of liver 
tests 
• 
Occurrence of a hepatic event considered to be an SAE 
Hepatic Monitoring Tests for a Hepatic Treatment Emergent Abnormality 
Hepatic Hematology 
Haptoglobin 
Hemoglobin 
 
Hematocrit 
Hepatic Coagulation 
RBC 
Prothrombin Time 
WBC 
Prothrombin Time, INR 
Neutrophils, segmented and bands 
 
Lymphocytes 
Hepatic Serologiesa 
Monocytes 
Hepatitis A antibody, total 
Eosinophils 
Hepatitis A antibody, IgM 
Basophils 
Hepatitis B surface antigen 
Platelets 
Hepatitis B surface antibody 
 
Hepatitis B Core antibody 
Hepatic Chemistry 
Hepatitis C antibody 
Total bilirubin 
Hepatitis E antibody, IgG 
Direct bilirubin 
Hepatitis E antibody, IgM 
Alkaline phosphatase 
 
ALT 
Anti-nuclear antibody 
AST 
Anti-actin antibody 
GGT 
CPK  
Anti-smooth muscle antibody 
 
Abbreviations: ALT = alanine aminotransferase; AST = aspartate aminotransferase; CPK = creatine phosphokinase; 
GGT = gamma-glutamyl transferase; Ig = immunoglobulin; INR = international normalized ratio; RBC = red 
blood cells; WBC = white blood cells. 
a Reflex/confirmation dependent on regulatory requirements and/or testing availability.