Clinical trial NCT03130439
Conditions: Breast Cancer
Keywords: Breast Cancer
Sponsor: Dana-Farber Cancer Institute
Start date: 2017-05-26; Completion date: 2022-01-30
Enrollment: 27 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This research study is studying a drug called Abemaciclib as a possible treatment for have metastatic triple-negative type of breast cancer.

Detailed description:
This research study is a Phase II clinical trial. Phase II clinical trials test the safety and effectiveness of an investigational intervention to learn whether the intervention works in treating a specific disease. "Investigational" means that the intervention is being studied.

The FDA (the U.S. Food and Drug Administration) has not approved abemaciclib as a treatment for any disease.

Some triple-negative breast cancers show expression of the Rb protein and are referred to as "Rb-positive." The Rb protein is important because it controls the way that cancer cells divide and grow. Drugs like abemaciclib work by changing the way that Rb functions. This can potentially stop cancer cells from dividing, and can also potentially lead to cancer cell death.

In this research study, the investigators are are looking to see how safe abemaciclib is and how well it will work to help people with triple-negative breast cancer that is Rb-positive.

Arms: arm 1: -Abemaciclib will be administered orally, twice daily on days 1 to 28
Interventions: intervention 1: Abemaciclib
Intervention descriptions: intervention 1: Abemaciclib (LY2835219) has been shown in vitro to be a selective ATP-competitive inhibitor of CDK4 and CDK6 kinase activity that prevents the phosphorylation and subsequent inactivation of the Rb tumor suppressor protein, thereby inducing G1 cell cycle arrest and inhibition of cell proliferation.

Protocol documents:
Prot_SAP_000.pdf: 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
2
SCHEMA
 
 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
3
TABLE OF CONTENTS
SCHEMA.........................................................................................................................................2
1.
OBJECTIVES......................................................................................................................6
1.1
Study Design............................................................................................................6
1.2
Primary Objectives...................................................................................................6
1.3
Secondary objectives ...............................................................................................6
2.
BACKGROUND .................................................................................................................6
2.1
Triple-Negative Breast Cancer ................................................................................6
2.2
Abemaciclib.............................................................................................................9
2.3
Rationale ................................................................................................................10
2.4
Correlative Studies Background ............................................................................11
3.
PARTICIPANT SELECTION...........................................................................................11
3.1
Inclusion Criteria ...................................................................................................11
3.2
Exclusion Criteria ..................................................................................................14
3.3
Inclusion of Women, Men, and Minorities............................................................16
4.
REGISTRATION PROCEDURES ...................................................................................16
4.1
General Guidelines for DF/HCC Institutions ........................................................16
4.2
Registration Process for DF/HCC Institutions.......................................................16
5.
TREATMENT PLAN........................................................................................................16
5.1
Treatment Regimen................................................................................................16
5.2
Pre-Treatment Criteria ...........................................................................................17
5.3
Agent Administration.............................................................................................18
5.4
General Concomitant Medication and Supportive Care Guidelines......................18
5.5
Criteria for Taking a Participant Off Protocol Therapy.........................................20
5.6
Duration of Follow Up...........................................................................................20
5.7
Criteria for Taking a Participant Off Study ...........................................................20
6.
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................21
6.1
Dose Adjustments and Delays ...............................................................................21
If a patient who, in the judgment of the investigator, is receiving clinical benefit 
from study..............................................................................................................22
6.2
Hematologic Toxicity ............................................................................................22
6.3
Non-hematologic Toxicity.....................................................................................22
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................23
7.1
Expected Toxicities................................................................................................23
7.2
Adverse Event Characteristics...............................................................................23
7.3
Expedited Adverse Event Reporting......................................................................24
7.4
Expedited Reporting to Hospital Risk Management .............................................24
7.5
Expedited Reporting to the Food and Drug Administration (FDA) ......................24
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
4
7.6
Expedited Reporting to t
7.7
Routine Adverse Event Reporting .........................................................................24
8.
PHARMACEUTICAL AGENT INFORMATION...........................................................25
8.1
Abemaciclib...........................................................................................................25
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................26
9.1
Clinical specimens collected..................................................................................26
9.2
Archival Tissue Specimens....................................................................................27
9.3
Circulating tumor DNA (ctDNA)..........................................................................29
9.4
Collection of Plasma Samples for Exploratory Biomarker Evaluations................30
9.5
Germline DNA.......................................................................................................30
9.6
Analysis of tissue biopsy specimens obtained at baseline and at C2D1................31
Specimens will be delivered to the Clinical Trials Core Laboratory for storage:.........................35
Coded laboratory specimens will be stored in the Clinical Trial Core Laboratory. These 
specimens will become the property of DFCI. Participants will be informed that their 
specimens may be used for research by investigators at DF/HCC and other approved 
collaborators. Shared specimens will be identified with a sample ID number; all patient 
identifying material will be removed.  Correlative studies described will be performed by 
Dr. Shom Goel in the Department of Cancer Biology at DFCI or relevant core facilities at 
DFCI. .................................................................................................................................35
10.
STUDY CALENDAR .......................................................................................................36
11.
MEASUREMENT OF EFFECT .......................................................................................38
11.1
Antitumor Effect – Solid Tumors ..........................................................................38
12.
DATA REPORTING / REGULATORY REQUIREMENTS...........................................44
12.1
Data Reporting.......................................................................................................45
12.2
Data Safety Monitoring..........................................................................................45
13.
STATISTICAL CONSIDERATIONS...............................................................................45
13.1
Study Design/Endpoints ........................................................................................45
13.2
Sample Size, Accrual Rate and Study Duration ....................................................46
13.3
Analysis of Primary Endpoints..............................................................................46
13.4
Analysis of Secondary Endpoints..........................................................................46
13.5
Reporting and Exclusions ......................................................................................48
All participants will be evaluable for toxicity from the time of their first treatment.....................48
14.
PUBLICATION PLAN .....................................................................................................48
15.
References..........................................................................................................................49
APPENDIX A:PERFORMANCE STATUS CRITERIA..............................................................51
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
5
APPENDIX B: USE OF STRECK TUBES ..................................................................................52
APPENDIX C: Inducers and Strong Inhibitors of Cyp3a4 or substrates of cyps with narrow 
therapeutic range................................................................................................................53
APPENDIX D: Archival tissue request instructions and form......................................................54
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
6
1. OBJECTIVES
1.1
Study Design
This is a single-arm, two-stage phase 2 study to evaluate the response to abemaciclib
monotherapy in patients with Rb-positive, triple negative metastatic breast cancer. The first
stage of the study includes 13 patients with Rb-positive, triple negative metastatic breast cancer. 
If in the first stage there is at least one response, accrual will continue to the second stage where 
an additional 25 patients will be enrolled. If there are at least 4 responses among the 38 patients, 
the regimen would be considered to be worthy of further study.
1.2
Primary Objectives
To evaluate the efficacy of abemaciclib, as defined by objective response rate (ORR) in patients 
with Rb-positive triple-negative metastatic breast cancer (ORR as confirmed Complete Response 
(CR) or Partial Response (PR) per Response Evaluation Criteria in Solid Tumors (RECIST) 
Version 1.1).
1.3
 Secondary objectives
1.3.1
To evaluate abemaciclib with respect to each of the following:
1) Progression-Free Survival (PFS)
2) Overall Survival (OS)
3) Disease control rate (DCR)
4) Clinical benefit rate (CBR)
To detect if there is a difference in clinical outcomes between patients with and without 
phospho-Rb reduction.
1.4
Exploratory objectives
To explore additional potential biomarkers in blood and tumor tissue samples that might predict 
response or resistance to abemaciclib.
To determine the association between genomic alterations in tumor DNA (mutations, copy 
number alterations, mutational burden) and RB expression and other tissue biomarkers by 
immunohistochemistry as well as response to abemaciclib. 
To determine if there are any changes in the T-cell phenotype during exposure to abemaciclib.
To explore changes in the microbiome induced by therapy comparing stool samples collected at 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
7
baseline, during treatment, and at the time of progression. Changes in microbiome will be 
compared to other trial endpoints. 
2. BACKGROUND
2.1
Triple-Negative Breast Cancer 
Breast cancer remains a major cause of cancer death for women globally. Indeed in the USA 
alone, more than 39,000 women succumbed to the disease is 2013 [1]. Breast cancer is 
comprised of a number of different subtypes, and the triple negative subtype (TNBC) currently 
presents the most significant clinical challenges [2] [3]. At present, the only available systemic 
therapies for patients with advanced TNBC are cytotoxic chemotherapies, and there are no 
targeted or biologic therapies approved to treat this disease [4].
Unfortunately, there are currently no therapeutic options for patients with advanced TNBC that 
has progressed after several lines of cytotoxic chemotherapy. Therefore, although TNBC 
constitutes approximately 20% of all breast cancers, it is the cause of almost half of all breast 
cancer deaths. Novel approaches to treating the disease are clearly needed.
Recently there has been increasing attention on the use of cell-cycle targeting agents in the 
treatment of breast cancer. In particular, inhibitors of the cyclin-dependent kinases CDK4 and 
CDK6 (CDK4/6 inhibitors) are the subjects of great interest, due to the fact that mammary 
epithelial cells (particularly those of the luminal lineage) are thought to depend on these CDKs 
for proliferation. In keeping with this, recent trials have confirmed striking efficacy of CDK4/6 
inhibitors in patients with estrogen-receptor positive breast cancers [5].
CDK4/6 inhibitors act by inhibiting the phosphorylation of the retinoblastoma tumor suppressor 
protein (RB). This in turn mitigates the expression of genes under the control of the E2F family 
of transcription factors, halting progression of tumor cells from the G1 to S phase of the cell 
cycle. As such, tumors that lack a functional RB protein (whether through gene deletion, 
mutation, or epigenetic silencing) are predicted to cycle in a CDK4/6-independent manner and 
thus be inert to the effects of CDK4/6 inhibition [6].
Traditionally, TNBC is associated with loss of function of RB and as such TNBC is not 
classically considered as a good candidate for response to CDK4/6 inhibition. However, it is 
clear that there is a subset of TNBC that retains intact RB protein and function. Depending on the 
methodology used, it is estimated that the rate of Rb loss in TNBC stands between 50-75% [6]. 
Therefore, there is potential that the remaining TNBC’s which retain RB function may exercise 
cell cycling in an RB-dependent fashion, and that CDK4/6 inhibitors could be an effective 
treatment strategy for these tumors.
Gene expression analysis from cohorts of TNBC’s have identified multiple different TNBC 
subtypes [7]. Notably, one subtype (the “Luminal Androgen Receptor” (LAR) subtype),
shows a strong androgen receptor driven pattern of gene expression, and LAR tumors typically 
express RB [8]. This group includes somewhere between 10-12% of TNBCs [9]. Recently, 
Traina et al presented results of a phase 2 study of an androgen receptor inhibitor in patients with 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
8
AR-positive metastatic TNBC (i.e. predominantly tumors of the LAR subtype). They observed 
an 8% response rate and a 16-week clinical benefit rate of 35% [9]. Given that this subset of 
TNBCs is typically RB-positive, there is a strong biological rationale to suggest that they might 
be susceptible to CDK4/6 inhibition. Furthermore, recent preclinical work suggests that triple-
negative cell lines of both the LAR and mesenchymal-stem like (MSL) subsets are sensitive to 
CDK4/6 inhibition in vitro [10]. Collectively, the LAR and MSL subtypes comprise 
approximately 20% of all TNBCs. 
Given the rationale for determining the effects of CDK4/6 inhibitors in TNBC with intact RB 
function, it is important to try and determine RB functionality in TNBCs. At present, there is no 
standardized way to do this. Various research groups have explored RB expression and/or 
functionality on TNBCs in the past. One approach has been to use immunohistochemistry (IHC) 
to the RB protein itself. Using this method, RB is detected in approximately 30-40% of all 
TNBCs [11, 12], using a definition of positive as “strong nuclear staining”. Another has been to 
measure tumor gene expression and apply signatures of “RB loss”. Interestingly, in at least one 
study such a signature correlated with the presence of loss of heterozygosity (LOH) for the RB 
locus, but did not correlate well with the presence of RB by IHC [13]. 
Notwithstanding these studies, there is currently limited data to support a particular method of 
determining RB functionality in TNBCs, and there is even less data to suggest that one modality 
would best predict a response to a CDK4/6 inhibitor. Knudsen’s laboratory has shown in a small 
number of TNBC explants that the presence of RB by IHC correlated with sensitivity to CDK4/6 
inhibition [14], and given the relative ease of this assay and the ability to apply it in real time to 
clinical care, this would appear to be a reasonable surrogate for RB functionality in TNBCs. 
In the present study, we have elected to use immunohistochemistry to screen tumors for the 
presence of the Rb protein. Immunohistochemistry has the following advantages:

RB presence by IHC has been shown to correlate with sensitivity to CDK4/6 
inhibitors in TNBC pre-clinically (ref 14)

Data describing RB-positivity rates by IHC are available from other cohorts 
(see above)

The assay can be performed rapidly and reproducibly
To optimize RB staining, we have worked with Dr. Joseph Geradts (pathologist, BWH), who has 
extensive experience in the study of RB in tumors by immunohistochemistry. We have elected to 
use the RB antibody clone G3-245 as this has been shown to yield reproducible, reliable RB 
staining in tissue sections when performed in the BWH IHC laboratory. Having selected this 
clone, we reviewed the RB staining characteristics (using clone G3-245) of a cohort of 180 
TNBCs in a tissue microarray curated by Dr. Nadine Tung and Dr. Stuart Schnitt at the BIDMC, 
with each case being represented on the microarray in triplicate. The results of this analysis have 
been formally presented (Patel et al, to ASCO 2017). Tumors were divided into 4 categories: 0% 
of tumor cells stained; 1-10% of tumor cells stained; 10-50% of tumor cells stained; >50% of 
tumor cells stained. Approximately 50% of tumors showed >10% of tumor cells positively 
stained. 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
9
We initially chose a cutoff of >10% to define RB positivity for the following reasons:

The percentage of RB positive tumors by IHC using this cutoff is similar to that seen in 
previous cohorts.

This cut off separated tumors into two clearly distinct cohorts with statistically different 
rates of Androgen Receptor positivity, tumor grade, and patient age (unpublished data). 
These are all features reported to be more common in RB-functional tumors when 
compared to RB non-functional tumors.  
However, after reviewing  the RB-positivity rates of the first 18 patients  the RB positivity rate  
was higher than that observed in the microarray (94% vs 50%). Two pathologists reviewed the 
stained sections for these cases and compared them to the initial microarray used to define RB 
cutoffs (Patel et al, ASCO 2017) and observed no differences in scoring techniques. 
The higher than expected rate of RB-positivity in the first 18 pre-screened metastatic patients 
might relate to differences in the populations studied in the initial microarray and our clinical 
study:

In the first 18 clinical cases, some of the archival primary tumors tested were of histologic 
subtypes expected to show high rates of RB expression (e.g. lobular carcinomas). Although 
these are typically ER-positive, patients with such cancers would still be eligible for the 
study as long as the most recent tumor tissue specimen from that patient had previously 
tested triple-negative (whether the primary in question nor a subsequent metastatic biopsy). 
These archival specimens, would be more likely to show Rb-expression than those in the 
microarray, which were all primary triple-negative ductal carcinomas. 

The microarray used to define staining cutoffs was enriched for patients with BRCA 
mutations, which showed a lower rate of RB expression than tumors from BRCA wild-type 
patients (Patel et al ASCO 2017). Indeed, the RB-positivity rate in BRCA wild-type tumors 
in the microarray was approximately 60 percent, rather than 50 percent as seen in the entire 
microarray population. Our initial 18 pre-screened patients were not enriched for BRCA 
mutation carriers, and might thus be expected to show a rate of RB-positivity higher than 50 
percent. 
The two reasons described above explain to some extent why the RB-positivity rate in our initial 
cohort was higher than 50 percent, as observed in the microarray. However, they are unlikely to 
explain the positivity rate as high as 94 percent. We thus also reviewed each case carefully to 
determine whether RB staining in our initial trial cohort might be “overcalling” RB-positivity.  
On review with pathologists (Dr. Geradts and Dr. Schnitt), we noted of the initial 18 cases pre-
screened, those with between 11-25% of tumor cells stained (i.e. in the lower range of 
“positive”) all showed very weak staining. Those with strong staining typically showed rates of 
staining of >50% of tumor cells. Given this, and the uncontrolled pre-analytic variables 
influencing immunohistochemical stains, we have elected to alter our definition of RB-positive 
from “ >10% of tumor cells staining” to “ >50% of tumor cells staining”. If this definition were 
applied to the initial microarray, the rate of RB positivity would be 42 percent amongst all 
TNBCs, and the rate in our initial cohort of 18 pre-screened patients would be 72%. Given the 
previously described differences in populations between the microarray and out clinical cohort, 
we believe that this cutoff represents a more reasonable choice to define “RB-positivity” in this 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
10
study.
Any patients enrolled on this trial prior to the RB positivity change from > 10% to > 50% being 
IRB approved and activated may remain on-study and continue treatment with Abemaciclib per 
protocol.
2.2
Abemaciclib
Abemaciclib is a selective and potent small molecule inhibitor of CDK4 and CDK6.
Abemaciclib demonstrates suitable physical and pharmacokinetic (PK) properties, an acceptable
toxicity profile in nonclinical species, and antitumor activity in multiple mouse models of human
cancer. Abemaciclib inhibits tumor growth in multiple human xenograft models including, but
not limited to, the following tumor types: 1) colorectal cancer, 2) glioblastoma multiforme,
3) acute myeloid leukemia, 4) non-small cell lung cancer, and 5) mantle cell lymphoma. The 
safety of abemaciclib (LY2835219) has been established in a phase 1, single-arm, dose-
escalation study in adult female patients with metastatic breast cancer. The most common grade 
3 adverse events (greater than 5% incidence) were neutropenia (33%), leukopenia (22%), 
abdominal pain (11%), diarrhea (6%) and fatigue (6%). Based on pharmacokinetic data, the 
recommended phase 2 dose was 200 mg po bid. Recently the results of the MONARCH-1 study 
were presented. In this study, women with advanced, ER-positive breast cancer that had 
progressed after 1 or 2 lines of systemic chemotherapy were treated with abemaciclib 
monotherapy. Strikingly, a 19.5% response rate was observed in this moderately pre-treated 
population (Dickler et al, ASCO 2016). This data suggests that abemaciclib can have meaningful 
clinical activity in RB-proficient tumors even in the context of chemotherapy resistance. 
The present study is a two-stage single arm phase 2 study of abemaciclib monotherapy in 
patients with metastatic RB-positive TNBC.The investigators will perform this study in 
compliance with the protocol, good clinical practice (GCP) and International Conference on 
Harmonization (ICH) guidelines, and applicable regulatory requirements.
Formally, abemaciclib refers to the free base, whereas LSN2813542 refers to abemaciclib
mesylate; however, abemaciclib has been used for uniformity throughout this protocol, except
when important for experimental clarity.
More information about the known and expected benefits, risks, and reasonably anticipated 
adverse events (AEs) of abemaciclib may be found in the Investigator’s Brochure (IB).  
Information on AEs expected to be related to the investigational product may be found in Section 
7 (Development Core Safety Information) of the IB. Information on serious adverse events 
(SAEs) expected in the study population independent of drug exposure and that will be assessed 
by the sponsor in aggregate, periodically during the course of the study, may be found in Section 
6 (Effects in Humans) of the IB.
2.3
Rationale
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
11
In cancer cells, genes that normally control the cell cycle are often dysregulated, leading to 
uncontrolled cellular proliferation [15]. The cyclin D-CDK4/6 holoenzyme controls G1 
restriction point via phosphorylation and deactivation of RB [16]. 
The clinical development of CDK4/6 inhibitors in breast cancer has largely been restricted to 
patients with ER-positive tumors, as the majority of TNBC’s is expected to lack functional RB 
and are thus not likely to respond to pharmacologic inhibition of CDK4/6. However, as described 
above a proportion of TNBCs are RB-proficient and in preclinical models have been shown to 
respond to CDK4/6 inhibition. The present study aims to determine the clinical efficacy and 
tolerability of abemaciclib in patients with RB-positive, advanced TNBC.
 
2.4
Correlative Studies Background
In this study, correlative scientific aims will be addressed by performing analysis of both 
archival patient specimens and specimens obtained during the course of the study. The 
overarching goal of these studies is to explore possible predictors of response to abemaciclib 
amongst RB-positive TNBC’s. Furthermore, studies will seek evidence of a pharmacodynamic 
response to abemaciclib therapy (through a tumor biopsy obtained on study) to determine if this 
correlates with response to therapy. 
The main candidate biomarker of tumor sensitivity to CDK4/6 inhibitors is the presence of 
intact, functional Rb [6]. At the time of screening patients for eligibility, therefore, we will 
perform immunohistochemistry to identify RB status on archival tumor tissue. Only patients that 
are RB-positive (greater than 50% of tumor cells staining positive) will be eligible for the study. 
Other than RB, there are no validated biomarkers for predicting response/resistance to CDK4/6 
inhibitors in breast cancer. Candidate markers of response include amplification of cyclin D1 and 
loss of the CDK4/6 inhibitor p16 [5], which are thought to render tumors more dependent on the 
CDK4/6 pathway. Conversely, possible predictors of resistance include amplification of cyclin 
E1/E2, overexpression of cyclin E, mutations in TP53, and heightened activity of 
phosphoinositide-dependent kinase 1. Finally, a failure of CDK4/6 inhibition to adequately 
achieve suppression of RB-phosphorylation, even in the presence of intact RB, is also predicted 
to be associated with a lack of response to therapy.
In this study, patients with biopsy-accessible disease will be required to undergo tumor biopsy 
prior to initiating therapy. In addition, patients will undergo a second biopsy after completion of 
1 cycle (28 days) of abemaciclib therapy. Analysis of pre-treatment biopsies (or archival tissue 
when pre-treatment biopsies are not available) will be performed to determine correlations with 
the predictive biomarkers listed above (and others) and response. Comparison of RB 
phosphorylation between baseline and C2D1 biopsies will be used to seek evidence of 
pharmacodynamic effect and explore its association with response. We will also perform 
exploratory genomic studies (whole exome sequencing) on the tumor tissue, as well as studies on 
fresh tumor tissue obtained at baseline aimed at determining therapeutic combinations that 
enhance the effects of CDK4/6 inhibitors in TNBC.   
Whole blood will be collected at the time of study entry for circulating tumor DNA (ctDNA). 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
12
From this sample, ctDNA will undergo whole exome sequencing (WES) in exploratory genomic 
studies to determine novel predictors of response to abemaciclib in TNBC.   Further details of all 
correlative studies are provided in section 9 of the protocol. 
The primary goal of this study is to determine the clinical efficacy of abemaciclib monotherapy 
in patients with advanced, “RB-positive” triple-negative breast cancer. For the purposes of the 
trial, we have defined RB positivity using an immunohistochemical assay – tumors in which 
greater than 50% of tumor cells stain positive for RB are deemed positive. In addition, to IHC, 
another important method for assessing RB function is through sequencing of DNA. Triple-
negative tumors can often harbor either mutations or deletions in RB1, which confer a loss of RB 
functionality. Thus, we also wish to determine whether RB1 alterations at a genomic level might 
predict the response of triple-negative breast cancers to abemaciclib. In order to do this, we will 
access DNA sequencing data (where possible) on tumors from all patients who participate in the 
pre-screening process. For the majority of these patients, DNA sequencing will have already 
been performed using the Dana-Farber Cancer Institute’s “Profile” targeted sequencing panel, as 
patients who sign consent for pre-screening in this study often also sign consent for other studies 
through which Profile testing is performed. Here, we propose to use this data to determine the 
impact of RB1 genomic alterations on response to abemaciclib, and also to explore correlations 
between RB1 genomic alterations and loss of RB staining on immunohistochemistry. 
Moreover, recent studies have implicated other genomic alterations which may also confer 
resistance to abemaciclib (for example, in TP53, PIK3CA, PTEN, and other genes) [20]. As 
such, we will also interrogate the Profile data these and other genomic alterations, as an 
exploratory analysis to determine genomic correlates of response to abemaciclib. 
Finally, recent data has suggested that CDK4/6 inhibitors can enhance anti-tumor immune 
responses not only through their effects on tumor cells, but also through direct effects on effector 
T lymphocytes (Deng, Cancer Discovery 2017; Schaer, Cell Reports 2018). As a result, we will 
also collect peripheral blood mononuclear cells from patients on study, with the intent of 
interrogating changes in T cell phenotype with abemaciclib therapy. 
The gut microbiota has been recognized as a modulator of immune system development. 
Healthy individuals have microbial populations in their intestinal tract that vary markedly in 
composition. The diversity of intestinal microbiota represents a significant challenge to the 
host’s immune defenses, which must balance immune tolerance of beneficial microbes with 
inflammatory responses against pathogens. Alterations in the gut microbiota and their resulting 
interactions with intestinal epithelieum and the host immune system are associated with many 
disease, including cancer. In addition, the identification of bacterial species associated with 
response could open new strategies to maximize the clinical benefit of cancer therapy through 
the modulation of gut microbiota.
3. PARTICIPANT SELECTION
3.1
Inclusion Criteria
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
13
3.1.1
Patients must have histologically or cytologically confirmed invasive breast 
cancer, which is recurrent, locally advanced, unresectable or metastatic. 
3.1.2
Patients must have at least one lesion that is not within a previously radiated field 
and that is measurable per RECIST version 1.1.  Bone lesions are not considered 
measurable.
3.1.3
Either the primary tumor and/or metastatic tumor must be triple-negative as 
defined below:

Hormone receptor status: the invasive tumor must be ER- and PR-
negative, or staining present in <1% by immunohistochemistry (IHC) 

HER2 status: the invasive tumor must be Human Epidermal Growth 
Factor Receptor 2 Negative (HER2-negative) by the ASCO CAP 
guidelines
In cases where both primary tumor and metastatic sample(s) have been tested for 
ER, PR, and HER2, then the triple-negative status of the tumor should be 
determined from the most recent sample available.
3.1.4
Either the primary tumor and/or the metastatic tumor must be RB positive as 
defined below:

RB status: the invasive tumor must have greater than 50% of tumor cells 
staining positive for RB.
3.1.5
 Prior Chemotherapy:

Patients may have received 1-3 prior systemic therapies for metastatic 
disease (note: for patients who have first developed recurrent/metastatic 
disease within 12 months of completing any (neo)-adjuvant therapy for 
triple-negative breast cancer, the (neo)-adjuvant therapy is counted as a 
prior line of therapy).

Patients must have been off treatment with myelosuppressive 
chemotherapy for at least 21 days or nonmyelosuppressive agents for 14 
days before registration.  Patients should also be adequately recovered (to 
baseline or grade 1) from acute toxicities of prior treatment except for 
residual alopecia and peripheral neuropathy. 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
14
3.1.6
Prior biologic therapy: Patients must have discontinued all biologic therapy at 
least 21 days before registration. 
3.1.7
Prior radiation therapy: Patients may have received prior radiation therapy in 
either the metastatic or early-stage setting.  Radiation therapy must be completed 
at least 7 days prior to study registration.
3.1.8
Patients on bisphosphonates or RANK-L inhibitors may continue receiving these 
therapies during study treatment. There is no washout period required between the 
last dose of these therapies and the start of abemaciclib. 
3.1.9
The patient has an Eastern Cooperative Oncology Group (ECOG) performance 
status 0-1 (ECOG scale provided as Appendix A)
3.1.10 Patients must have normal organ and marrow function as defined below:

Absolute neutrophil count 1500/mm3

Platelets 100,000/mm3

Hemoglobin 8 g/dL 
Note: Patients may receive erythrocyte transfusions to achieve this 
hemoglobin level at the discretion of the investigator. Initial treatment 
must not begin earlier than the day the after the erythrocyte infusion

Total Bilirubin 1.5x the upper limit of normal (ULN) or ≤ 2.0 x the 
upper limit of normal (ULN) in patients with documented Gilbert’s 
syndrome.

Serum creatinine 1.5 mg/dL OR calculated GFR 60mL/min

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 
3 times the upper limit of normal.  For patients with documented 
liver metastases, AST/ALT ≤ 5.0 times the upper limit of normal. 
3.1.11 Female subjects of childbearing potential must have a negative serum pregnancy 
test at screening. Women of childbearing potential are defined as those who have 
not been surgically sterilized and have had a menstrual period in the past year
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
15
3.1.12 The patient must be  18 years old
3.1.13 Capable of understanding and complying with the protocol and has signed the 
informed consent document.
3.1.14 Able to swallow study drug.
3.1.15 Sexually active patients (male and female) must use medically acceptable 
methods of contraception during the course of the study and for 3 months after 
completion of study treatment. If a woman becomes pregnant or suspects she is 
pregnant while participating in this study, she should inform her treating 
physician immediately.  While on the study and for 3 months after final drug 
administration, women may not breast-feed. 
3.1.16 Confirmed availability of formalin-fixed, paraffin-embedded (FFPE) tumor tissue 
3.1.17 Patients with tumor that is felt to be accessible to biopsy must be willing to 
provide tissue from a newly obtained core biopsy of a tumor lesion at baseline.  
Biopsies will be obtained up to 1 week (7 days) prior to initiation of treatment on 
Cycle 1, Day 1.  Patients who undergo an attempted research biopsy procedure for 
the purpose of this protocol, and in whom inadequate tissue is obtained, are not 
required to undergo a repeat biopsy in order to continue on protocol.  
3.2
Exclusion Criteria
A patient will be excluded from the study if he or she meets any of the following criteria:
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
16
3.2.1
Received a prior CDK4/6 inhibitor.
3.2.2
Undergone major surgery within 14 days of the initial dose of study drug
3.2.3
Received another investigational agent (defined as any agent/device that has not 
received regulatory approval for any indication) within 14 days of the first dose of 
study drug for a nonmyelosupressive agent, or 21 days of the first dose of study 
drug for a myelosuppressive agent.  
3.2.4
Has any severe concurrent disease, infection, or comorbid condition that renders 
the patient inappropriate for enrollment in the opinion of the investigator.
3.2.5
Has an active bacterial infection (requiring IV antibiotics at the time of initiating 
study treatment), fungal infection, or detectable viral infection. Patients with 
known HIV infection are excluded given the potential for interactions between 
antiretroviral agents and abemaciclib, and the potential for increased risk of life-
threatening infection with therapy that is myelosuppressive. Patients with known 
Hepatitis B or Hepatitis C infection are excluded only if there is evidence of 
active infection (detectable Hepatitis B surface antigen, detectable Hepatitis C 
RNA) 
3.2.6
Documented brain metastases that are untreated, symptomatic, or require therapy 
to control symptoms. Participants with previously diagnosed brain metastases are 
eligible if they have completed treatment at least 7 days prior to trial registration, 
are neurologically stable, and have recovered from effects of radiotherapy or 
surgery.  

Any corticosteroid use for brain metastases must have been discontinued 
without the subsequent appearance of symptoms for ≥2 weeks before the 
first study drug.  

Treatment for brain metastases may have included whole brain 
radiotherapy, radiosurgery, or a combination as was deemed appropriate 
by the treating physician.

Patients who meet the above criteria and are clinically stable on anti-
convulsant medication are eligible only if their anti-convulsant does not 
alter hepatic cytochrome P450 activity in a way that might interfere with 
metabolism of abemaciclib (see Appendix C). 
3.2.7
Pregnant women are excluded from this study because of the potential for 
teratogenic effects.  
3.2.8
Lactating women are excluding from the study.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
17
3.2.9
Individuals with a history of a second malignancy are ineligible except for the 
following circumstances: individuals with a history of other malignancies are 
eligible if they have been disease-free for at least 5 years and are deemed by the 
investigator to be at low risk for recurrence of that malignancy. Individuals with 
the following cancers are eligible if they are diagnosed and have completed 
treatment within the past 5 years: cervical cancer in situ, and non-melanoma 
cancer of the skin.  Patients with other cancers diagnosed within the past 5 years 
and felt to be at low risk of recurrence should be discussed with the principal 
investigator to determine eligibility.
3.2.10 Have received any live vaccination within 28 days of first dose of study drug. 
3.2.11 Has a personal 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), or sudden 
cardiac arrest. 
3.3
Inclusion of Women, Men, and Minorities
Both men and women of all races and ethnic groups are eligible for this trial.
4. REGISTRATION PROCEDURES
4.1
General Guidelines for DF/HCC Institutions
Institutions will register eligible participants in the Clinical Trials Management System (CTMS) 
OnCore. Registrations must occur prior to the initiation of protocol therapy. Any participant not 
registered to the protocol before protocol therapy begins will be considered ineligible and 
registration will be denied.
An investigator will confirm eligibility criteria and a member of the study team will complete the 
protocol-specific eligibility checklist.
Following registration, participants may begin protocol therapy. Issues that would cause 
treatment delays should be discussed with the overall Principal Investigator (PI). If a participant 
does not receive protocol therapy following registration, the participant’s registration on the 
study must be canceled. Registration cancellations must be made in OnCore as soon as possible. 
4.2
Registration Process for DF/HCC Institutions
DF/HCC Standard Operating Procedure for Human Subject Research Titled Subject Protocol 
Registration (SOP #: REGIST-101) must be followed.  
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
18
5. TREATMENT PLAN
5.1
Treatment Regimen
Abemaciclib will be administered orally, twice daily on days 1 to 28 of a 28 day cycle as 
described in Table 5.1. Reported adverse events and potential risks are described in Section 7.  
Appropriate dose modifications are described in Section 6.  No investigational or commercial 
agents or therapies other than those described below may be administered with the intent to treat 
the participant's malignancy.
TABLE 5.1
TREATMENT REGIMENS/DOSING SCHEDULE 
Regimen
Period/Cycle
Dose Day
Abemaciclib
Treatment/28-day cycle
200 mg PO Q12H on Days 1-28
Abbreviations: PO=Orally
Q12H=every 12 (±2) hours.
The investigator or his/her designee is responsible for the following:

explaining the correct use of the drug and planned duration of each individual’s 
treatment to the patient/site personnel/legal representative,

verifying that instructions are followed properly,

maintaining accurate records of study drug dispensing and collection
For assessing compliance with treatment, the participant will be requested to maintain a 
medication diary of each dose of medication. The medication diary will be returned to clinic staff 
at the end of each cycle.
Patients will be instructed to contact the investigator as soon as possible if they have a complaint 
or problem with the study drug so that the situation can be assessed.
5.2
Pre-Treatment Criteria
On day 1 of cycle 1, blood will be drawn for complete blood count (CBC), liver function tests, 
urea, electrolytes, and creatinine.  Results must be reviewed by the treating physician and meet 
the criteria below: 

Absolute neutrophil count 1500/mm3

Platelets 100,000/mm3

Hemoglobin 8 g/dL

Total Bilirubin 1.5x the upper limit of normal (ULN)

Serum creatinine 1.5 mg/dL OR calculated GFR 60mL/min

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 
3 times the upper limit of normal.  For patients with documented 
liver metastases, AST/ALT ≤ 5.0 times the upper limit of normal. 
On day 1 of subsequent cycles, blood will be drawn for complete blood count (CBC), liver 
function tests, urea, electrolytes, and creatinine.  Results must be reviewed by the treating 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
19
physician and meet the criteria below: 

Absolute neutrophil count 1000/mm3

Platelets 100,000/mm3

Total Bilirubin 1.5x the upper limit of normal (ULN)

Serum creatinine 1.5 mg/dL OR calculated GFR 60mL/min

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 
3 times the upper limit of normal.  For patients with documented 
liver metastases, AST/ALT ≤ 5.0 times the upper limit of normal. 
5.3
Agent Administration
5.3.1
Abemaciclib will be supplied as capsules for oral administration. The capsules 
should be stored at room temperature according to the range provided on the 
product label and not opened, crushed, or dissolved. Investigators should instruct 
patients to store the capsules in the original package and in a location inaccessible 
to children. Clinical study materials will be labeled according to country 
regulatory requirements.
5.3.2
Abemaciclib will be taken orally every 12 (± 2) hours on Days 1 through 28 of a 
28-day cycle, for a total of 56 doses per cycle. Patients should not consume food 
beginning 1 hour before and ending 1 hour after taking abemaciclib. During all 
cycles, abemaciclib should be taken at approximately the same times each day. If 
a patient misses or vomits a dose, that dose should be omitted.
5.3.3
 A patient may continue to receive study drug until she meets 1 or more of the 
specified reasons for discontinuation (as described in Section 5.5.)
5.4
General Concomitant Medication and Supportive Care Guidelines
With the exceptions provided in the sections below, no other chemotherapy, experimental
medications, other anticancer therapy, immunotherapy, hormonal cancer therapy, surgery for 
cancer, or experimental medications will be permitted while patients are on study
treatment.  Radiation while on study treatment will be permitted after discussion with the PI. 
The results from an in vitro human recombinant cytochrome P450 (CYP) phenotyping study
indicate that oxidative metabolism of abemaciclib is primarily catalyzed by CYP3A4. However,
the extent of oxidative metabolism responsible for the systemic clearance of abemaciclib in
humans is unknown. Based on these in vitro findings, grapefruit juice as well as
inducers (for example, phenytoin and carbamazepine) and strong inhibitors of CYP3A should
be substituted or avoided if possible (Appendix C). 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 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
20
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. 
In vitro studies in primary cultures ofhuman hepatocytes indicate that abemaciclib might inhibit 
the metabolism of CYP2B6 substrate drugs in vivo. Based on this finding, sensitive substrates of 
CYP2B6 such as bupropion and efavirenz should be substituted or avoided if possible.
5.4.1
Supportive Care
Patients should receive full supportive care to maximize quality of life. Patients will receive
supportive care based on the judgment of the treating physician. If it is unclear whether a
therapy should be regarded as supportive care, the investigator should consult the principal 
investigator (PI).
5.4.2
Growth Factor Therapy
Growth factors may be administered in accordance with American Society of Clinical Oncology
Guidelines [17]. Dosing of abemaciclib must be suspended if the administration of growth 
factors is required and must not be restarted within 48 hours of the last dose of growth factors 
having been administered.  When restarted, the dose of abemaciclib must be reduced by 1 dose 
level if a dose reduction for the specific event necessitating the use of the growth factors has not 
already occurred.
5.4.3
Supportive Management for Diarrhea
At enrollment, patients should receive instructions on the management of diarrhea. In the event 
of diarrhea, supportive measures should be initiated as early as possible.  These include the 
following: 

At the sign of loose stools, patients should initiate anti-diarrheal therapy (for example, 
loperamide) and notify the investigator/site for further instructions and appropriate 
follow-up.

Participants should be encouraged to drink fluids (for example, 8 to 10 glasses of clear 
liquids per day)

Site personnel should assess response within 24 hours

If diarrhea does not resolve with anti-diarrheal therapy within 24 hours to either baseline 
or Grade 1, study drug should be held until diarrhea is resolved to baseline or grade 1. 

If diarrhea resolves to baseline or grade 1, participants should resume dosing as outlined 
in Section 6.3.2 and Table 6.1. 
In severe cases of diarrhea, measuring neutrophil counts and body temperature and 
proactively managing diarrhea with antidiarrheal agents is recommended (page 16). In 
addition, patients with ≥ grade 3 diarrhea should be reviewed by a treating physician once per 
week until the diarrhea has resolved to ≤ grade 1. 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
21
If diarrhea is severe (requiring IV rehydration) and/or associated with fever or severe
neutropenia, broad-spectrum antibacterial therapy, including coverage for enteric (gram-negative 
and
anaerobic) bacteria. If neutropenic fever persists for 5 days despite broad-spectrum antibacterial
therapy, empiric antifungal therapy should be initiated. If diarrhea persists after antibiotic 
treatment, testing for Clostridium difficile is indicated as antibiotic therapy might precipitate C. 
difficile infection. 
Patients with severe diarrhea or any grade of diarrhea associated with severe nausea or vomiting 
should be carefully monitored and given IV fluid (IV hydration) and electrolyte replacement. In 
these patients, electrolytes and renal function should be monitored regularly during the period of 
IV hydration. 
5.5
Criteria for Taking a Participant Off Protocol Therapy.
Duration of therapy will depend on individual response, evidence of disease progression and 
tolerance. In the absence of treatment delays due to adverse event(s), treatment may continue 
until one of the following criteria applies:
Disease progression
Intercurrent illness that prevents further administration of treatment
Unacceptable adverse event(s)
Participant demonstrates an inability or unwillingness to comply with the oral 
medication regimen and/or documentation requirements
Participant decides to withdraw from the protocol therapy
General or specific changes in the participant's condition render the participant 
unacceptable for further treatment in the judgment of the treating investigator
Participants will be removed from the protocol therapy when any of these criteria apply.  The 
reason for removal from protocol therapy, and the date the participant was removed, must be 
documented in the case report form (CRF) and in the clinical trial management system, OnCore. 
Alternative care options will be discussed with the participant.
In the event of unusual or life-threatening complications, treating investigators must immediately 
notify the overall PI, Dr. Sara Tolaney at 617-632-2335. 
5.6
Duration of Follow Up
Participants should remain in long-term follow-up with disease status and overall survival status 
checked once every 6 months until death.  There are no long-term follow-up visits required.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
22
Participants removed from protocol therapy for unacceptable adverse event(s) will be followed 
until resolution or stabilization of the adverse event.
5.7
Criteria for Taking a Participant Off Study
Participants will be removed from study when any of the following criteria apply:

Lost to follow-up

Withdrawal of consent for data submission

Death
The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the case report form (CRF) and updated with the relevant Off Treatment/Off 
Study information in OnCore. 
6. DOSING DELAYS/DOSE MODIFICATIONS
6.1
Dose Adjustments and Delays
Table 6.1 Toxicity Dose Adjustments and Delays for Abemaciclib
Toxicity Type
Toxicity Profile and Severity
Dose
Hematologic Toxicity
Sections 6.2 and 5.4.2
Requiring administration of 
growth factors, regardless of 
severity 
(Growth factors use permitted 
according to ASCO Guidelines)
Dose MUST be 
suspended
for at least 48 hours 
after the last dose of 
blood cell growth 
factors was
administered and until
toxicity resolves to at 
least Grade 2.
Dose MUST be reduced
by 1 dose level unless
already performed for
incidence of toxicity that
lead to the use of growth
factor.
Hematologic Toxicity
Section 6.2
Grade 3
Dose MUST be 
suspended
until toxicity resolves 
to at least Grade 2.
Dose MAY be reduced
by 1 dose level -
investigator’s discretion.
Hematologic Toxicity
Section 6.2
Recurrent Grade 3
Dose MUST be 
suspended
until toxicity resolves 
to at least Grade 2.
Dose MUST be reduced
by 1 dose level.
Hematologic Toxicity
Section 6.2
Grade 4
Dose MUST be 
suspended
until toxicity resolves 
to at least Grade 2.
Dose MUST be reduced
by 1 dose level.
Non-hematologic Toxicity
(except diarrhea and increased 
AST/ALT)
Section 6.3
Persistent or recurrent Grade 2 
that does not resolve with 
maximal supportive measures 
within 7 days to baseline or 
Grade 1
Dose MUST be 
suspended
until toxicity resolves 
to either baseline or 
Grade 1.
Dose MUST be reduced
by 1 dose level
Non-hematologic Toxicity
(except diarrhea and increased 
AST/ALT)
Section 6.3
Grade 3 or 4
Dose MUST be 
suspended
until toxicity resolves 
to either baseline or 
Dose MUST be reduced
by 1 dose level.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
23
Grade 1.
Diarrhea
Sections 6.3.2 and 5.4.3
Grade 2
Dose MUST be
suspended if toxicity 
does not resolve 
within 24 hours to ≤ 
Grade 1.
Dose reduction is not 
required.
Diarrhea
Sections 6.3.2 and 5.4.3
Persistent or recurrent Grade 2 
that does not resolve with 
maximal supportive measures 
within 24 hours to at least 
Grade 1
Dose MUST be
suspended until 
toxicity
resolves to at least 
Grade 1.
Dose MUST be reduced
by 1 dose level -
investigator's discretion.
Diarrhea
Sections 6.3.2 and 5.4.3
Diarrhea recurs despite 
maximal supportive measures 
after resuming same dose level 
after initial Grade 2 diarrhea
Dose MUST be 
suspended
until toxicity resolves 
to at least Grade 1.
Dose MUST be reduced
by 1 dose level.
Diarrhea
Sections 6.3.2 and 5.4.3
Requires hospitalization or 
Grade 3 or 4
Dose MUST be 
suspended
until toxicity resolves 
to at least Grade 1.
Dose MUST be reduced
by 1 dose level.
Hepatic Toxiciy (AST and/or 
ALT)
Section 6.3.4
Persistent or recurrent Grade 2 
(> 3.0-5.0 x ULN) 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
Dose MUST be 
suspended until 
toxicity resolves to 
baseline or Grade 1.
Dose MUST be reduced 
by 1 dose level.
Hepatic Toxiciy (AST and/or 
ALT)
Section 6.3.4
Grade 3 (>5.0-20.0 x ULN) 
AND Total Bilirubin > 2.0 x 
ULN, in the absence of 
cholestasis.
Discontinue 
Abemaciclib.
N/A
Hepatic Toxiciy (AST and/or 
ALT)
Section 6.3.4
Grade 4 (>20.0 x ULN)
Discontinue 
Abemaciclib.
N/A
Abbreviation: ASCO = American Society of Clinical Oncology.
Note: MAY = per the investigator’s clinical judgment; SHOULD = not mandatory but highly recommended;
MUST = mandatory.
6.1.1
Abemaciclib Dose Adjustments
If a patient who, in the judgment of the investigator, is receiving clinical benefit from study
therapy requires further dose reduction than is outlined in Table 6.1, then the investigator
must discuss with the PI prior to any further dose reduction. For patients requiring dose
reduction(s), re-escalation to a prior dose level is permitted only after consultation with the
PI.
A cycle is defined as the planned treatment interval of 28 days plus any subsequent delay prior to
start of the next cycle. A delay in the start of a cycle due to holidays, weekends, bad weather, or
other unforeseen circumstances will be permitted up to 7 days and not counted as a protocol
deviation. No more than 56 doses of abemaciclib should be dispensed for each 28-day cycle.
Dose omissions are allowed within a cycle. If a patient requires omission of more than 25% of
doses during a cycle for tolerability, then treatment may continue if the investigator determines
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
24
the patient is receiving clinical benefit.
Table 6.1.1
Dose Adjustments of Abemaciclib 
Dose Adjustment
Oral Dose
Frequency
0
200 mg
Every 12 hours
1
150 mg
Every 12 hours
2
100 mg
Every 12 hours
3
50 mg
Every 12 hours
6.2
Hematologic Toxicity
Hematologic toxicities including neutropenia, leukopenia, anemia, and thrombocytopenia have 
been observed in patients treated with abemaciclib, and causality has been established. Patients 
should be monitored closely for signs of infection, anemia, and bleeding. Dose adjustment may 
be considered for hematological toxicity ≥Grade 3. Hematological toxicities must resolve to 
baseline or at least Grade 2 prior to the start of each cycle. Blood cell growth factors are only to 
be used in a manner consistent with American Society of Clinical Oncology (ASCO) guidelines. 
Fever or infection in the presence of severe neutropenia should be managed promptly with 
broad-spectrum antibacterial therapy, including coverage for enteric (gram-negative and 
anaerobic) bacteria. If neutropenic fever persists for 5 days despite broad-spectrum antibacterial 
therapy, empiric antifungal therapy should be initiated. Anemia and thrombocytopenia should be 
treated supportively and if necessary with red cell or platelet transfusions.
Tables 6.1 and 6.1.1 provide further detail on abemaciclib dose modification for hematologic 
toxicity. 
6.3
Non-hematologic Toxicity
6.3.1
Dose adjustment for other non-hematologic toxicity (other than diarrhea)
Before the start of each cycle, non-hematologic toxicity must be evaluated.  All non-hematologic 
toxicities (except alopecia, diarrhea and fatigue) possibly related to abemaciclib must resolve to 
either baseline or at least Grade 1.
If a patient experiences persistent or recurrent Grade 2 non-hematologic toxicity (except 
diarrhea; refer to Section 6.3.2) possibly related to abemaciclib that does not resolve with 
maximal supportive measures within 7 days to either baseline or Grade 1, then dosing may be 
suspended (until the toxicity resolves to either baseline or at least Grade 1) and the dose of 
abemaciclib may be reduced as outlined in Table 6.1.1.
If a patient experiences ≥ Grade 3 non-hematologic toxicity possibly related to abemaciclib, then
dosing must be suspended (until the toxicity resolves to either baseline or at least Grade 1) and
the dose of abemaciclib must be reduced as outlined in Table 6.1.1.
 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
25
6.3.2
Diarrhea
A patient experiencing diarrhea requiring hospitalization (irrespective of grade, that is, requiring 
intravenous [IV] rehydration) or severe diarrhea (Grade 3 or 4) must have study treatment 
suspended (until the toxicity resolves to either baseline or Grade 1) and should have the study 
drug dose reduced by 1 dose level as outlined in Table 6.1.
If a patient experiences persistent or recurrent diarrhea that does not resolve with maximal
supportive measures within 24 hours to either baseline or at least Grade 1, then study treatment 
should be suspended (until the toxicity resolves to either baseline or at least Grade 1) and the 
dose of study drug may be reduced by 1 dose level as outlined in Table 6.1.1 at the discretion of 
the investigator. If the participant resumes treatment at the same dose level and diarrhea recurs 
despite maximal supportive measures, the dose of study drug must be reduced by 1 dose level as 
outlined in table 6.1.1. 
6.3.3
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 are at the discretion of the treating provider, but none are stipulated by 
this protocol. 
6.3.4
General Guidance for Hepatic Monitoring
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation are considered 
expected with the use of abemaciclib. See Table 6.1 for dose modification guidance. Hepatic 
monitoring should depend upon the severity and persistence of the observed laboratory test 
abnormalities. If a study patient experiences elevated AST/ALT 5.0xULN and elevated total 
bilirubin 2.0xULN, or AST/ALT 8.0xULN, liver tests (including ALT, AST, TBL, direct 
bilirubin, gamma-glutamyl transferase (GGT), and creatinine phosphokinase (CPK), should be 
repeated within 3 to 5 days to confirm the abnormality and to determine if it is increasing or 
decreasing. If the abnormality persists or worsens, clinical and laboratory monitoring should be 
initiated by the investigator, based on the hepatic monitoring tests below: 
Table 6.3.4: Hepatic Monitoring Tests for a Hepatic Treatment Emergent Abnormality.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
26
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
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of reported and/or potential AEs (Section 7.1) and the characteristics of an 
observed AE (Section 7.2) will determine whether the event requires expedited reporting in 
addition to routine reporting
7.1
Expected Toxicities
The most common AEs considered related to abemaciclib were headache (14%),
nausea (11%), diarrhea (8%), and vomiting (8%). 
7.2
Adverse Event Characteristics

CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be 
downloaded from the CTEP web site 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
27
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.

Attribution of the AE:
-
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.
7.3
Expedited Adverse Event Reporting 
7.3.1
Investigators must report to the Overall PI any serious adverse event (SAE) that occurs 
after the initial dose of study treatment, during treatment, or within 30 days of the last 
dose of treatment on the local institutional SAE form. 
7.3.2
DF/HCC Expedited Reporting Guidelines
Investigative sites within DF/HCC will report AEs directly to the DFCI Office for 
Human Research Studies (OHRS) per the DFCI IRB reporting policy. 
7.4
Expedited Reporting to Hospital Risk Management
Participating investigators will report to their local Risk Management office any participant 
safety reports or sentinel events that require reporting according to institutional policy. 
7.5
Expedited Reporting to the Food and Drug Administration (FDA)
The Overall PI, as study sponsor, will be responsible for all communications with the FDA. The 
Overall PI will report to the FDA, regardless of the site of occurrence, any serious adverse event 
that meets the FDA’s criteria for expedited reporting following the reporting requirements and 
timelines set by the FDA. 
7.6
SAEs will be reported to
 on a MedWatch form as soon as possible and no later than 24 
hours of the Investigator and/or Institution receiving notification of any serious adverse event 
experienced by a patient participating in the study and receiving study drug.  Fax SAEs to the 
local safety representative using the following Fax number:  866-644-1697.
7.7
Routine Adverse Event Reporting
All Grade 2 or higher Adverse Events must be reported in routine study data submissions to the 
Overall PI on the toxicity case report forms.  AEs reported through expedited processes (e.g., 
reported to the IRB, FDA, etc.) must also be reported in routine study data submissions.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
28
8. PHARMACEUTICAL AGENT INFORMATION
A list of the adverse events and potential risks associated with the investigational agent 
administered in this study can be found in Section 7.1.
8.1
Abemaciclib
8.1.1
Description
Abemaciclib, (IUPAC: N-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-
fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine) , or  LY2835219, is 
a potent and selective inhibitor of CDK4 and CDK6 with IC50 of 2 nM and 10 nM, 
respectively.  It has a molecular weight of 602.7 and it chemical formula is 
C28H36F2N8O3S.  The half-life is 17-38 hours. 
8.1.2
Storage and Stability
Abemaciclib needs to be stored in the original package. Abemaciclib needs to be kept at 
room temperature (approximately 15-30 degrees Celsius wherever possible). 
8.1.3
Handling
Qualified personnel, familiar with procedures that minimize undue exposure to 
themselves and the environment, should undertake the preparation, handling, and safe 
disposal of the chemotherapeutic agent in a self-contained and protective environment. 
8.1.4
Availability
Abemaciclib will be provided for clinical trial use as 50mg of abemaciclib with 
excipients in hypromellose capsules as individual patient supply packaged in bottles. 
Storage conditions are described in the medication label.
8.1.5
Preparation
Participants will be provided with an adequate supply of abemaciclib for self-
administration at home, including instructions for administration, until at least their next 
scheduled study visit. Participants will receive abemaciclib on an outpatient basis. The 
investigator shall provide the participant with instructions for abemaciclib administration 
according to the protocol.
The investigator or responsible site personnel must instruct the participant or caregiver to 
take abemaciclib as per protocol. Abemaciclib will dispensed to the participant by 
authorized site personnel only. All dosages prescribed to the participant and all dose 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
29
changes during the study must be recorded on the Dosage Administration Record CRF.
8.1.6
Administration
Abemaciclib is administered orally. 
Compliance should be assessed by the investigator and/or study personnel at each 
participant visit and information provided by the participant and/or caregiver. Records of 
study medication used, dosages administered, and intervals between visits and the 
completion of the study will be captured in the Drug Accountability Form. This 
information must be captured in the source document at each participant visit.
8.1.7
Ordering
Abemaciclib will be provided to patients 
 
8.1.8
Accountability
The investigator, or a responsible party designated by the investigator, should maintain a 
careful record of the inventory and disposition of the agent using the NCI Drug 
Accountability Record Form (DARF) or another comparable drug accountability form.  
(See the NCI Investigator’s Handbook for Procedures for Drug Accountability and 
Storage).
All drug supplies are to be used only for this protocol, and not for any other purpose.
8.1.9
Destruction and Return
The study drug supply will be destroyed according to the DFCI Institutional Pharmacy 
SOP.
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES
9.1
Clinical specimens collected
The following clinical specimens will be collected from patients on this study:
a) Archival tumor tissue if available (paraffin-embedded)
b) Blood at baseline (for collection of plasma, germline DNA and ctDNA).
c) Blood at each time of restaging (for collecting ctDNA)
d) Blood at C1D15, C2D1, C3D1, and at progression (for plasma)
e) Tumor biopsy material obtained at time of study entry (if accessible disease) – both fresh 
frozen and formalin-fixed, paraffin-embedded
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
30
f)
Tumor biopsy material obtained at time of C2D1 (if accessible disease) – both fresh 
frozen and formalin-fixed, paraffin-embedded
g) Blood at time of progression for collection of ctDNA
h) Stool at baseline, on treatment, toxicity, and EOT/PD
Sample
Type
Visit
Archival Tissue 
FFPE 
Tumor
Screening
Fresh Tissue
FFPE and Frozen
 Tumor
  Screening and Cycle 2 day 1
cfDNA
Blood
Screening, Restaging, and Progression
Plasma for isolation of 
exosomes
Blood
Screening, C1D15, C2D1, C3D1, and 
Progression
Germline DNA
Blood
Screening
PBMCs
Blood
Screening, C2D1, C3D1, and 
Progression
Stool
Stool
Screening, Between C2D1-C3D1, 
grade ≥ 2 diarrhea (optional), and 
EOT/PD
9.2
Archival Tissue Specimens
9.2.1
Collection of Archival specimen(s)
Archival tissue will be collected at screening to determine tumor RB status and potential 
eligibility for the trial.  RB status will be determined using immunohistochemistry on 
formalin-fixed, paraffin embedded tissue.  
The staining will be done using Rb antibody clone G3-245 using a protocol developed in the 
BWH clinical immunohistochemistry laboratory (CLIA conditions). A BWH pathologist (Dr 
Stuart Schnitt will review the stained slides and results will be reported to the study team. To 
be considered RB positive, the invasive tumor must have greater than 50% of tumor cells 
staining positive for RB. The expected turnaround time between receipt of tissue (slides or 
blocks) and delivery of the staining result is expected to be approximately 7-14 days. 
We are requesting that 1 FFPE block or 15 unstained slides and 1 representative H&E stained 
slide containing tumor tissue be provided from each patient’s definitive surgery or metastatic 
biopsy.  The tissue requisition provided in (Appendix D) should be completed and submitted 
 
 
 
 
 
 










DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
35
as well, as deemed appropriate and informative based on the state of the immune profiling literature 
at the time of correlative science performance: CD4, FOXP3, CD127.
9.6.3
Site Performing Correlatives:
DFCI CIO 
9.7
Analysis of tissue biopsy specimens obtained at baseline and at C2D1
9.7.1
Guidelines for Tissue Acquisition and Biopsies of Metastatic Lesions
9.7.1.1 Collection of Specimen(s)
Tissue specimens will be collected from recurrent or metastatic lesions using standard 
institutional procedures. The amount of tissue collected will follow the guidelines listed 
below. If a participant has more than one site of disease, only one site needs to be biopsied 
in order to go on to the study and the site is left to the discretion of the patient and their 
treating physician. Core biopsies are preferred over fine needle aspirates when both are 
technically feasible. However, fine needle aspirates are acceptable and may be used for the 
baseline tissue sample. Participants who undergo a research biopsy procedure for the 
purpose of this protocol, and in whom inadequate tissue is obtained, are still eligible and 
are not required to undergo a repeat biopsy in order to enter the study.  Research biopsies 
can be waived, with PI approval, in participants who do not have easily accessible disease.
For the baseline biopsy, ideally five core biopsies will be obtained and processed as follows:

First core will be placed in 10% neutral buffered formalin and then made into a formalin 
fixed paraffin embedded (FFPE) block.

Second core will be frozen in OCT

Third core will be placed in 10% neutral buffered formalin and then made into an FFPE 
block.**

Fourth core will be frozen in OCT**

Fifth core will be placed in DMEM
**If a biopsy yields less than five cores, the PI may opt to have the 3rd or 4th core placed in DMEM 
to send to the lab of Dr. Peter Sicinski. This can be discussed with Dr. Goel in advance of the 
procedure. 
For the C2 biopsy, ideally five core biopsies will be obtained and processed as follows:

First core will be placed in 10% neutral buffered formalin and then made into a formalin 
fixed paraffin embedded (FFPE) block.

Second core will be frozen in OCT

Third core will be placed in 10% neutral buffered formalin and then made into an FFPE 
block.

Fourth core will be frozen in OCT
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
36

Fifth core will be frozen in OCT
Guidelines for biopsy from various metastatic sites can be found below.
Breast: A goal of 3-6 core biopsy specimens will be obtained using standard institutional 
guidelines for a diagnostic core biopsy of a breast mass.
Skin/chest wall: A goal of 3 x 4-mm punch biopsies will be obtained.
Lymph node: A goal of 3-6 core biopsy specimens will be obtained using an 18-gauge 
needle.
Liver: A goal of 3-6 core biopsy specimens will be obtained using an 18-gauge needle.
Lung: Because of the risk of pneumothorax associated with core needle biopsies of lung 
nodules, no core biopsies of lung nodules will be performed on this protocol, unless they are 
clinically indicated.
Bone: Because the yield of malignant tissue from bone biopsies tends to be relatively low, 
if a participant has another accessible site of disease (i.e. skin, lymph node, liver), that site 
should be biopsied preferentially. If bone is the only biopsy-accessible site, then a goal of 
3-6 core biopsy specimens will be obtained using an 11-13 gauge needle.
Pleural Fluid: A goal of 500 cc of pleural fluid will be obtained with a standard thoracentesis 
procedure, with or without image guidance, according to the clinical judgment of the treating 
physician and clinician performing the procedure. Less than the goal amount is acceptable, 
and should be based upon the clinical judgment of the Investigator and the clinician 
performing the procedure. If more than the goal amount of fluid is obtained, then the entire 
specimen (with the exception of what is needed for clinical purposes, if applicable) will be 
stored in the tissue bank.
Ascites fluid: A goal of 500 cc of ascites fluid will be obtained with a standard paracentesis 
procedure, with or without image guidance, according to the clinical judgment of the treating 
physician and clinician performing the procedure. Less than the goal amount is acceptable, 
and should be based upon the clinical judgment of the Investigator and the clinician performing 
the procedure.  If more than the goal amount of fluid is obtained, then the entire specimen 
(with the exception of what is needed for clinical purposes, if applicable) will be stored in the 
tissue bank.
9.7.1.2 Site(s) Performing Correlative Study
Dana-Farber Cancer Institute
9.7.1.3 Risks of Research Biopsy and Procedures for Minimizing Risk
Potential risks according to site are:
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
37
Breast (core biopsy):
• Likely: local discomfort and minor bleeding.
• Less likely: moderate or major bleeding, need for blood transfusion, hospitalization 
due to bleeding or other complications, infection, pneumothorax, damage to adjacent 
organs.
Skin/chest wall (punch biopsy):
• Likely: local discomfort and minor bleeding
• Less likely: moderate or major bleeding, or infection
Lymph node, liver, or bone (core needle biopsy):
• Likely: local discomfort and minor bleeding
• Less likely: moderate or major bleeding, need for blood transfusion, hospitalization 
due to bleeding or other complications, infection, damage to adjacent organs. 
Additional risks may be present if intravenous conscious sedation is required
Pleural fluid (thoracentesis):
• Likely: local discomfort and minor bleeding
• Less likely: moderate or major bleeding, need for blood transfusion, hospitalization 
due to bleeding or other complications, infection, pneumothorax, damage to 
adjacent organs
Ascites fluid (paracentesis):
• Likely: local discomfort and minor bleeding
• Less likely: moderate or major bleeding, need for blood transfusion, hospitalization  
due to bleeding or other complications, infection, bowel perforation or damage to 
adjacent organs. In order to minimize the risk of a biopsy, only qualified personnel 
will perform these procedures.
Prior to the procedure, the physician performing the procedure will discuss the risks with 
each study participant, answer any questions, and obtain separate procedure consent. Patients 
will be evaluated for comorbidities or concomitant medications that may increase the risk of 
potential complications. For biopsies of lesions that are not superficial and clearly palpable, 
imaging studies such as CT or ultrasound will be used to guide the biopsy in order to 
minimize the risk of  damage to adjacent structures. After lymph node biopsies, patients will 
be observed a minimum of 2 hours (range 2-4 hours) after the procedure, or according to 
standard institutional guidelines. After liver biopsies, patients will be observed a minimum of 
4 hours (range 4-6 hours) after the procedure, or according to standard institutional guidelines. 
Less than the goal quantity of tissue is accepted for each type of biopsy, and will be left to 
the clinical judgment of the physician performing the procedure.
9.7.1.4 Risks of Anesthesia
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
38
Local Anesthesia
All biopsy procedures require local anesthesia using lidocaine, xylocaine, or related 
compounds. There is a small risk of an allergic reaction associated with these drugs. 
In order to minimize the risk of local anesthesia, only qualified personnel will perform 
the biopsy procedure. Patients will be queried if they have had previous allergic 
reactions to local anesthetics.
Intravenous Conscious Sedation
Certain biopsy procedures, such as lymph node, liver, or bone biopsies, may require 
intravenous conscious sedation (IVCS). IVCS is a minimally depressed level of 
consciousness that retains the patient's ability to maintain a patent airway independently and 
continuously and respond appropriately to physical stimulation and verbal commands.
The risks of intravenous conscious sedation include: inhibition of the gag reflex and 
concomitant risk of aspiration, cardiopulmonary complications (myocardial infarction, 
cardiac arrhythmias, hypoxemia), and allergic reactions to the sedative or analgesic 
medications. These risks are small but real; for example, in a prospective study of 14,149 
patients undergoing IVCS during upper gastrointestinal endoscopies, the rate of immediate 
cardiopulmonary events was 2 in 1000. The 30-day mortality was 1 per 2,000 cases. In this 
study, there was a strong association between lack of monitoring and use of high-dose 
benzodiazepines with adverse outcomes. There was also an association between the use of 
local anesthetic sprays to the oropharynx and the development of pneumonia. In order to 
minimize the risk of intravenous conscious sedation, only qualified personnel will be 
responsible for conscious sedation. A minimum of two individuals will be involved in the 
care of patients undergoing conscious sedation-the physician performing the biopsy 
procedure, and the individual (M.D. or R.N.) who monitors the patients and his/her response 
to both the sedation and the procedure, and who is capable of assisting with any supportive 
or resuscitative measures. The room where the procedure utilizing IVCS takes place will have 
adequate equipment to provide supplemental oxygen, monitor vital signs, and maintain an 
airway should this be necessary. An emergency cart will also be immediately accessible to 
the room where the procedure is to take place, and emergency support services will be 
available on page. Patients will be screened and evaluated for their fitness to undergo 
conscious sedation by a trained physician. Patients with active cardiac disease are excluded 
from this study. No local anesthetic spray to the oropharynx will be necessary, given that 
endoscopy is not a planned procedure. Following the procedure, patients will be observed 
closely in the recovery room for a minimum of 2 hours.
General Anesthesia
Because of the higher risk of general anesthesia compared with local anesthesia or intravenous 
conscious sedation, biopsies that would require general anesthesia in order to be performed 
are not permitted on this protocol, unless they are being done for clinical reasons, and excess 
tissue that otherwise would have been discarded is then banked for the purpose of this 
 
 
 
 
 
 




DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
40
Tumor tissue obtained from the baseline biopsy will be used to examine candidate markers of 
sensitivity or resistance to abemaciclib therapy, based on available preclinical and clinical data.
a) DNA: extracted DNA will be subject to whole exome sequencing as an exploratory analysis. 
Specifically, we will determine copy number for cyclin D1 and cyclin E1/2, and also 
mutational data for TP53 (approximately 60-80% of TNBCs contain a TP53 mutation). We 
will determine whether the rate of response is lower in tumors cyclin E amplification, or 
tumors with TP53 mutations. Exploratory analyses will also be performed to determine other 
novel predictors of response to abemaciclib. 
b) RNA: extracted RNA will be used to determine expression levels of genes including but not 
limited to, cyclin D1, cyclin E1, cyclin E2, CDKN2A, CDKN1A, CDKN1B, CDK2, TSC2, 
and PDK1. In each case, will provide descriptive statistics comparing response rates between 
tumors with higher or lower levels of expression of these genes. 
c) Protein: Baseline core biopsies will be stained for androgen receptor (AR) and RB. We 
expect that most, but not all, tumors will be AR-positive. We will determine response rates in 
patients with AR-positive vs AR-negative tumors. We will also document the concordance 
rate between RB positivity in archival and metastatic samples.
d) Fresh tissue: when at least 3-5 cores are obtained from baseline biopsies, 1 will be delivered 
fresh to the laboratory of Dr Peter Sicinski (DFCI). The Sicinski lab team will dissociate this 
tissue to isolate cancer cells and test novel methods to enhance the efficacy of CDK4/6 
inhibition in TNBC cells.
A portion of the baseline biopsy will have RNA extracted. This RNA will then be subjected to 
analysis of gene expression by Affymetrix microarray and/or using the Nanostring nCounter 
platform. Tumors will be classified according to published algorithms to determine their 
individual TNBC subtype. We expect that the majority (but not all) of the RB-positive tumors 
will fall into the “Luminal Androgen Receptor” or “Mesenchymal Stem Like” subtypes, and as 
an exploratory analysis will determine if tumors within this subtype have a higher rate of 
response to abemaciclib. Additionally, in patients who consent, RNA extracted will be used to 
quantify the expression of several genes, including (but not limited to) those used to classify 
tumors into PAM50 subtypes[18], those identifying specific TNBC subtypes [19][8], and those 
that contribute to a signature of RB proficiency or deficiency. This will allow us to depict the 
landscape of gene expression for all tumors in this study, and also to determine in an exploratory 
fashion the correlation (or lack thereof) between RB positivity by IHC and “Rb-proficiency” by 
gene expression. The Nanostring assay will be performed at the BWH Center for Advanced 
Molecular Diagnostis (CAMD). Some portion of de-identified patient clinical information will 
be shared with Nanostring Technologies, with patient permission, in exchange for the kits 
needed to perform this assay. 
Analysis of paired baseline and C2D1 biopsies
a) Phospho-RB: FFPE tissue from baseline and C2D1 biopsies will be stained for p-RB 
(ser780) using protocols established at the Brigham and Women’s Hospital. Sections will 
be scored manually and the percentage of positive tumor cells will be recorded – a 
minimum of 200 cells will be counted. A tumor H score will be calculated. We will 
determine whether (i) baseline RB phosphorylation level predicts response to abemaciclib 
and (ii) whether suppression of RB phosphorylation after commencement of therapy is 
associated with an increased response rate. 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
41
b) FFPE tissue from baseline and C2D1 biopsies will be stained for proteins involved in the 
regulation of cell proliferation in the lab of Kornelia Polyak, MD, PhD at Dana-Farber 
Cancer Institute.
9.8
Research Stool Collection
9.8.1
Collection
All stool samples will be collected by each patient at home using a home-based, self-collection 
and mail method for stool that has been proven to provide nearly equivalent metagenomic and 
metatranscriptomic data to state-of-the-art fresh-frozen sample-collection protocol. 
Patient questionnaires are included in the kits. The questionnaires should be completed at the 
time of stool collection and mailed back along with the sample. Stool samples and questionnaires 
that are not collected at the protocol-specified collection time points will not be protocol 
violations.
Given that this procedure was added to the study mid-way through accrual, only new participants 
will be approached to participant in this correlative study. If a patient doesn’t complete a baseline 
collection, the series may not continue as this collection is essential to analysis. 
9.8.2
Handling and shipping of stool specimens
All kits will be provided to the patients at a clinic visit or will be mailed to their home address. 
Patients will also be provided with a mailer in which to return the sample. All samples will be 
shipped by the patient at ambient temperature to the Breast Tumor Immunology Lab (BTIL, 
Elizabeth Mittendorf, MD, PhD) where they will be processed and stored until batch shipment 
for analysis.
9.8.3
Analysis
We will quantify microbiome features from amplicon, metagenome, metatranscriptome using 
established pipelines to identify strain-level taxonomic, functional gene, transcriptional, and 
microbially-mediated metabolite profiles associated with patients with Rb+ TNBC treated with 
abemaciclib.
9.8.4
Sites performing correlative analysis
Dana-Farber Breast Tumor Immunology Lab (BTIL, Elizabeth Mittendorf, MD, PhD)
Microbiome Dx
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
42
10. STUDY CALENDAR
Baseline evaluations are to be completed within 28 days prior to registration unless otherwise 
specified.  Laboratory assessments, including pregnancy test, are to be completed within 14 days 
of registration.  In the event that the participant’s condition is deteriorating, laboratory 
evaluations should be repeated within 48 hours prior to initiation of the next cycle of therapy.
Assessments must be performed prior to administration of any study agent. Study assessments 
and agents should be administered within + 3 days of the protocol-specified date, unless 
otherwise noted.  Tumor assessments must be completed within + 7 days of the protocol-
specified date.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
43
Cycle 1
Cycle 2
Cycle 3
Cycle 4+
Screening
D1
D15
D1
D28
D1
D1
D28
30  
Day 
FUVk
Follow
-Up
Informed consent
X
Demographics
X
Medical history, 
including past lines 
of therapy
X
Physical exam
X
X
X
X
X
X
X
Vital signsa
X
X
X
X
X
X
X
Height
X
Weight
X
X
X
X
X
X
Performance status
X
X
X
X
X
X
Concurrent Meds
X
X
X
X
X
X
X
AE Evaluation
X
X
X
X
X
X
X
CBC w/diff
X
X
X
X
X
X
X
Serum chemistryb
X
X
X
X
X
X
X
Serum cystatin Cc
X
X
X
X
X
X
X
B-HCG
Xd
Blood for ctDNAe
X
X
X
Blood for genomic 
sequencing control
X
Blood for plasma 
exosome analysisf
X
X
X
X
X
Blood for germline 
DNAg
X
Blood for PBMCs
X
X
X
X
Archival tumor 
tissue
X
Research stool 
collection and 
questionnairem
X
X
X
X
Research Biopsy
X
X
EKGh
X
Tumor assessmentsi
X
X
X
Xj,k
Abemaciclib
X--------------------------------------------------------X
Survival Follow-up
X
Abemaciclib: 200mg po bid for 28 day cycles.
 
a.
Vital signs to include: diastolic and systolic blood pressure, heart rate and temperature
b.
Chemistry to include: Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, 
chloride, creatinine, glucose, potassium, total protein, SGOT [AST], SGPT [ALT], sodium.
c.
Cystatin C should be performed each time serum chemistries are performed, as abemaciclib is known to 
compete for tubular secretion of creatinine, causing an increase in serum creatinine which does not 
reflect changes in renal function. Cystatin C allows for more accurate assessment of renal function in 
this context. 
d.
Serum pregnancy test (women of childbearing potential as defined in the eligibility section)
e.
ctDNA to be drawn at C1D1, each restaging vist and at time of progression/30 Day FUV (Streck)
f.
Plasma to be drawn at C1D1, C1D15, C2D1, C3D1 and at time of progression/30 Day FUV (EDTA)
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
44
g.
Blood drawn at C1D1 for collection of germline DNA (Streck)
h.
EKGs to be done at Screening and if clinically indicated
i.
Tumor assessments will be performed every 2 cycles (8 weeks) for the first 6 cycles (6 months) and 
then every 3 cycles (12 weeks) going forward.  
j.
Tumor assessments should be obtained at 30 day FUV if not done within the previous 8 weeks
k.
Follow-up survival (disease status and overall status) follow-up evaluation – a separate clinic visit is 
not required, this can be done over the phone every six months.  
l.
30 Day FUV to be completed within 30 days of last dose of abemaciclib
m. Research stool collection to occur at baseline, between C2D1 and C3D1, at the time of grade ≥ 2 
diarrhea, and at the end of treatment or progression, whichever occurs first.
11. MEASUREMENT OF EFFECT
11.1
Antitumor Effect – Solid Tumors
A baseline tumor evaluation must be performed within 4 weeks before patient begins study
treatment. Restaging scans will be performed every 8 weeks. 
Response and progression will be evaluated in this study using the international criteria proposed 
by the New Response Evaluation Criteria in Solid Tumors (RECIST): Revised RECIST
Guideline (version 1.1; Eisenhauer et al. 2009). Changes in the largest diameter (unidimensional 
measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph 
nodes are used in the RECIST criteria.
11.1.1 Definitions
Evaluable for Target Disease response.  Only those participants who have measurable 
disease present at baseline, have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for target disease response.  These 
participants will have their response classified according to the definitions stated below.  
(Note:  Participants who exhibit objective disease progression prior to the end of cycle 1 
will also be considered evaluable.)
Evaluable Non-Target Disease Response.  Participants who have lesions present at 
baseline that are evaluable but do not meet the definitions of measurable disease, have 
received at least one cycle of therapy, and have had their disease re-evaluated will be 
considered evaluable for non-target disease.  The response assessment is based on the 
presence, absence, or unequivocal progression of the lesions. 
11.1.2 Disease Parameters
Tumor lesions/lymph nodes will be categorized at baseline as measurable or non-
measurable. Measurable disease is defined by the presence of at least 1 measurable 
lesion.
11.1.2.1
Measurable
Tumor lesions: Measured in at least 1 dimension (longest diameter in the plane of 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
45
measurement is to be recorded) with a minimum size of:

10 mm by computed tomography (CT) or magnetic resonance imaging (MRI)
scan (slice thickness 5 mm)

10 mm caliper measurement by clinical exam (non-measurable lesions if cannot
be accurately measured with calipers)

20 mm by chest X-ray.
Malignant lymph nodes: To be considered pathologically enlarged and measurable, a 
lymph node must be ≥15 mm in short axis when assessed by CT scan (CT scan slice 
thickness recommended to be 5 mm).
11.1.2.2
Nonmeasurable
All other lesions, including small lesions (longest diameter <10 mm or pathological 
lymph nodes with ≥10 to <15 mm short axis) as well as truly non-measurable lesions. 
Lesions considered truly non-measurable include: leptomeningeal disease, ascites 
pleural/pericardial effusions, lymphangitis cutis/pulmonis, inflammatory breast disease, 
lymphangitis involvement of skin or lung, abdominal masses/abdominal organomegaly 
identified by physical exam that is not measureable by reproducible imaging techniques.
11.1.2.3
Special Considerations for Lesion Measurability
Bone lesions:

Bone scan, PET scan or plain films are not considered adequate imaging
techniques to measure bone lesions.

Lytic bone lesions or mixed lytic-blastic lesions, with identifiable soft tissue
components, that can be evaluated by cross sectional imaging techniques such as 
CT or MRI, can be considered measurable lesions if the soft tissue component
meets the definition of measurability.

Blastic bone lesions are non-measurable.
Cystic Lesions: 

Simple cysts should not be considered as malignant lesions (neither
measurable nor nonmeasurable)

Cystic lesions thought to represent cystic metastases can be considered as
measurable lesions, if they meet the definition of measurability. If non-cystic 
lesions are presented in the same patients, these are preferred for selection as
target lesions.
Lesions with Prior Local Treatment:

Tumor lesions situated at a previously irradiated area, or in an area subjected
to other loco-regional therapy, are non-measurable unless there has been
demonstrated progression in the lesion.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
46
11.1.3 Target lesions 
When more than 1 measurable lesion is present at baseline, all lesions up to a maximum 
of 5 lesions total (and a maximum of 2 lesions per organ) representative of all involved 
organs should be identified as target lesions and will be recorded an measured at baseline. 
Non-nodal target lesions should be selected on the basis of their size (lesions with the 
longest diameter), be representative of all involved organs, and can be reproduced in 
repeated measurements. Measurable lymph nodes are target lesions if they meet the 
criteria of a short axis of ≥15 mm by CT scan. All measurements are to be recorded in the 
case record form (CRF) in millimeters (or decimal fractions of centimeters [cm]).
11.1.4 Non-target lesions.  
All other lesions (or sites of disease) are identified as non-target lesions (chosen based on 
their representativeness of involved organs and the ability to be reproduced in repeated 
measurements) and should be recorded at baseline. Measurement of these lesions are not 
required but should be followed as ‘present,’ ‘absent,’ or in rare cases ‘unequivocal 
progression.’ In addition, it is possible to record multiple non-target lesions involving the 
same organ as a single item on the CRF (for example, multiple liver metastases recorded 
as 1 liver lesion).
Lymph nodes with short axis ≥10 mm but <15 mm should be considered non-target 
lesions. Nodes that have a short axis <10 mm are considered non-pathological and are not 
recorded or followed.
11.1.5 Methods for Evaluation of Disease
All measurements should be taken and recorded in metric notation using a ruler, calipers, 
or a digital measurement tool.  All baseline evaluations should be performed as closely as 
possible to the beginning of treatment and never more than 4 weeks before the beginning 
of the treatment.
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up. Imaging-based 
evaluation is preferred to evaluation by clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam. 
An adequate volume of a suitable contrast agent should be given so that the metastases 
are demonstrated to best effect and a consistent method is used on subsequent 
examinations for any given patient. If prior to enrollment it is known a patient is not able 
to undergo CT scans with IV contrast due to allergy or renal insufficiency, the decision as 
to whether a non-contrast CT or MRI (with or without IV contrast) should be used to 
evaluate the patient at baseline and follow-up should be guided by the tumor type under 
investigation and the anatomic location of the disease.
Clinical lesions. Clinical lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes) and ≥10 mm in diameter as 
assessed using calipers (e.g., skin nodules).  In the case of skin lesions, documentation by 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
47
color photography, including a ruler to estimate the size of the lesion, is recommended. 
When lesions can be evaluated by both clinical exam and imaging, imaging evaluation 
should be undertaken since it is more objective and may be reviewed at the end of the 
study.
Chest x-ray.  Chest CT is preferred over chest X-ray when progression is an important 
endpoint. Lesions on chest X-ray may be considered measurable if they are clearly 
defined and surrounded by aerated lung.
Conventional CT and MRI. CT scan is the best currently available and reproducible 
method to measure lesions selected for response assessment. Measurability of lesions on 
CT scan is based on the
assumption that CT slice thickness is ≤5 mm. When CT scan have slice thickness >5 mm, 
the minimum size for a measurable lesion should be twice the slice thickness. MRI is also 
acceptable in certain situations (for example, for body scans). If there is concern about 
radiation exposure at CT, MRI may be used instead of CT in selected instances.
Pet Scan (FDG-PET, PET CT): PET is not recommended for lesion assessment. If a new 
lesion is found by PET, another assessment must be done by CT, unless the PET CT is of 
diagnostic quality. If CT is done to confirm the results of the earlier PET scan, the date of 
progression must be reported as the earlier date of the PET scan.
Ultrasound. Ultrasound should not be used to measure lesion size. Ultrasound 
examinations cannot be reproduced in their entirety for independent review at a later date 
and, because they are operator dependent, it cannot be guaranteed that the same technique 
and measurements will be taken from one assessment to the next. If new lesions are 
identified by ultrasound in the course of the study, confirmation by CT or MRI is 
advised.
Endoscopy, Laparoscopy. The utilization of these techniques for objective tumor 
evaluation is not advised. However, such techniques may be useful to confirm complete 
pathological response when biopsies are obtained or to determine relapse in trials where 
recurrence following complete response (CR) or surgical resection is an endpoint.
Tumor markers. Tumor markers alone cannot be used to assess response. If markers are 
initially above the upper normal limit, they must normalize for a participant to be 
considered in complete clinical response.  Specific guidelines for both CA-125 response 
(in recurrent ovarian cancer) and PSA response (in recurrent prostate cancer) have been 
published [JNCI 96:487-488, 2004; J Clin Oncol 17, 3461-3467, 1999; J Clin Oncol 
26:1148-1159, 2008].  In addition, the Gynecologic Cancer Intergroup has developed 
CA-125 progression criteria which are to be integrated with objective tumor assessment 
for use in first-line trials in ovarian cancer [JNCI 92:1534-1535, 2000].
Cytology, Histology. These techniques can be used to differentiate between partial 
responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor 
types, such as germ cell tumors, where known residual benign tumors can remain).
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
48
When effusions are known to be a potential adverse effect of treatment (for example,
with certain taxane compounds or angiogenesis inhibitors), the cytological confirmation 
the neoplastic origin of any effusion that appears or worsens during treatment can be 
considered if the measurable tumor has met criteria for response or stable disease (SD) in 
order to differentiate between response (or SD) and progressive disease (PD).
Bone Scan: If lesions measured by bone scan are reported at baseline, it is necessary to 
repeat the bone scan when trying to identify a complete response (CR) or partial response 
(PR) in target disease or when progression in bone is suspected.
11.1.6 Response Criteria
11.1.6.1
Evaluation of Target Lesions:
Complete Response (CR):  Disappearance of all target lesions.  Any pathological 
lymph nodes (whether target or non-target) must have reduction in short axis to <10 
mm.
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters.
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of 
target lesions, taking as reference the smallest sum on study (this includes the 
baseline sum if that is the smallest on study).  In addition to the relative increase of 
20%, the sum must also demonstrate an absolute increase of at least 5 mm. The 
appearance of one or more new lesions is also considered progressions.
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on 
study.
Not Evaluable: When an incomplete radiologic assessment of target lesions is 
performed or there is a change in the method of measurement from baseline that 
impacts the ability to make a reliable evaluation of response.
11.1.6.2
Evaluation of Non-Target Lesions
Complete Response (CR):  Disappearance of all non-target lesions and normalization 
of tumor marker level.  All lymph nodes must be non-pathological in size (<10 mm 
short axis).
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits.
Progressive Disease (PD):  Appearance of one or more new lesions and/or 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
49
unequivocal progression of existing non-target lesions. 
Not Evaluable: When a change in method of measurement from baseline occurs and 
impacts the ability to make a reliable evaluation of response.
11.1.6.3
Evaluation of New Lesions
The finding of a new lesion should be unequivocal (i.e. not due to difference in 
scanning technique, imaging modality, or findings thought to represent something 
other than tumor (for example, some ‘new’ bone lesions may be simply healing or 
flare of pre-existing lesions). However, a lesion identified on a follow-up scan in an 
anatomical location that was not scanned at baseline is considered new and will 
indicate PD. If a new lesion is equivocal (because of small size etc.), follow-up 
evaluation will clarify if it truly represents new disease and if PD is confirmed, 
progression should be declared using the date of the initial scan on which the lesion 
was discovered.
11.1.6.4
Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of the study 
treatment until the earliest of objective progression or start of new anticancer therapy, 
taking into account any requirement for confirmation. The patient’s best overall 
response assignment will depend on the findings of both target and non-target disease 
and will also take into consideration the appearance of new lesions. The Best Overall 
Response will be calculated via an algorithm using the assessment responses provided 
by the investigator over the course of the trial.
11.1.6.5
Time Point Response
It is assumed that at each protocol-specified time point, a response assessment occurs. 
(When no imaging/measurement is done at all at a particular time point, the patient is 
not evaluable (NE) at that time point.) Table 11.1.4.5 provides a summary of the 
overall response status calculation at each time point for patients who have 
measurable disease at baseline.
Table 11.1.4.5            Time Point Response: Patients with Target (+/- Nontarget) Disease
Target 
Lesions
Non-Target 
Lesions
New 
Lesions
Overall 
Response
Best Overall Response when 
Confirmation is Required
CR
CR
No
CR
>4 wks Confirmation
CR
Non-CR/Non-
PD
No
PR
CR
Not evaluated
No
PR
>4 wks Confirmation
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
50
PR
Non-CR/Non-
PD/not 
evaluated
No
PR
SD
Non-CR/Non-
PD/not 
evaluated
No
SD
Documented at least once >4 
wks from baseline
PD
Any
Yes or No
PD
Any
PD*
Yes or No
PD
Any
Any
Yes
PD
no prior SD, PR or CR
Not all evaluated
Non-PD
No
SD
Abbreviations: CR = complete response; PR = partial response; SD = stable disease; PD = progressive disease;
NE = inevaluable.
*      In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as disease 
progression.
11.1.7 Duration of Response
Duration of overall response:  The duration of overall response is measured from the 
time measurement criteria are met for CR or PR (whichever is first recorded) until the 
first date that recurrent or progressive disease is objectively documented (taking as 
reference for progressive disease the smallest measurements recorded since the treatment 
started, or death due to any cause. Participants without events reported are censored at the 
last disease evaluation).
Duration of overall complete response: The duration of overall CR is measured from the 
time measurement criteria are first met for CR until the first date that progressive disease 
is objectively documented, or death due to any cause. Participants without events 
reported are censored at the last disease evaluation.
Duration of stable disease:  Stable disease is measured from the start of the treatment 
until the criteria for progression are met, taking as reference the smallest measurements 
recorded since the treatment started, including the baseline measurements. 
11.1.8 Response Review
The study will use the DF/HCC Tumor Imaging Metrics Core (TIMC) for central protocol 
measurements.
12. DATA REPORTING / REGULATORY REQUIREMENTS
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
51
12.1Data Reporting
12.1.1 Method
The Office of Data Quality (ODQ) will collect, manage, and perform quality checks on 
the data for this study.
12.1.2 Responsibility for Data Submission
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 
data forms to the Office of Data Quality in accordance with DF/HCC SOPs.
12.2
Data Safety Monitoring
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor 
toxicity and accrual data from this study. The committee is composed of clinical 
specialists with experience in oncology and who have no direct relationship with the 
study. Information that raises any questions about participant safety will be addressed 
with the overall PI and study team.
The DSMC will review each protocol up to four times a year or more often if required to 
review toxicity and accrual data. Information to be provided to the committee may 
include: up-to-date participant accrual; current dose level information; DLT information; 
all grade 2 or higher unexpected adverse events that have been reported; summary of all 
deaths occurring with 30 days of intervention for Phase I or II protocols; for gene 
therapy protocols, summary of all deaths while being treated and during active follow-
up; any response information; audit results, and a summary provided by the study team. 
Other information (e.g. scans, laboratory values) will be provided upon request. 
12.3
Collaborative Research and Future Use of Data and Specimens
Tissue, blood, bodily fluids, and other materials derived from these will be collected in this study 
to analyze genes, DNA, RNA, proteins and cells for the study’s correlative endpoints and potential 
future research, utilizing new types of biomarker testing as it becomes available. 
These samples and any data generated as a part of these clinical trials may be used for future 
research studies and may be provided to collaborating investigators both within and outside of the 
DF/HCC for either correlative endpoints or secondary use. Samples and data may be shared with 
outside non-profit academic investigators, as well as with for-profit pharmaceutical investigators 
or commercial entities, with whom we collaborate. When samples or data are sent to collaborators 
and when any research is performed on them, all information will be identified with a code, and 
will not contain any PHI, such as name, birthday, or MRNs. 
In order to allow the greatest amount of research to be performed on the specimens and 
information generated as a part of this trial, researchers in this study may share results of genetic 
sequencing with other scientists. De-identified specimen or genetic data may be placed into one 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
52
of more publicly-accessible scientific databases, such as the National Institutes of Health’s 
Database for Genotypes and Phenotypes (dbGaP). The results from the correlative research on 
this study will be shared with these public databases. Through such databases, researchers from 
around the world will have access to de-identified samples or data for future research. More 
detailed information, beyond the public database, may only be accessed by scientists at other 
research centers who have received special permission to review de-identified data 
13. STATISTICAL CONSIDERATIONS   
13.1
Study Design/Endpoints
This is a single-arm, two-stage phase 2 study to evaluate the response to abemaciclib
monotherapy in patients with Rb-positive, triple negative metastatic breast cancer. 
13.1.1 Primary objective and endpoint
To evaluate the efficacy of abemaciclib, as defined by objective response rate (ORR) in patients 
with Rb-positive triple-negative metastatic breast cancer (ORR as confirmed Complete Response 
(CR) or Partial Response (PR) per Response Evaluation Criteria in Solid Tumors (RECIST) 
Version 1.1).
13.1.2  Secondary efficacy endpoints 
1) Progression-Free Survival (PFS)
2) Overall Survival (OS).
3) Disease control rate (DCR)
4) Clinical benefit rate (CBR)
13.2
Sample Size, Accrual Rate and Study Duration
This is a single-arm, two-stage phase 2 study in order to determine the efficacy of abemaciclib 
monotherapy in patients with metastatic RB-positive TNBC. There is no data for response to 
abemaciclib in patients with TNBC who are RB-positive.   A response rate of 20% would be of 
clinical interest.  A true response rate of 5% would not be of clinical interest. We have used a 
Simon optimal two-stage model with a one-sided type I error of 10% and type II error of 0.1 
(90% power) to detect the difference between null (5%) and alternative (20%) objective response 
rates. The first stage of the study includes 12 patients with metastatic triple-negative breast 
cancer which is RB-positive. As of June 2018, it was discovered that a participant without 
measurable disease was registered and treated on this trial. As such, this participant is considered 
unevaluable in relation to the primary endpoint. Therefore, we plan to enroll a total of 13 
participants to the first stage, with the expectation that 12 will be evaluable for response. The 
statistical plan will otherwise remain unchanged. If in the first stage there is at least one 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
53
response, accrual will continue to the second stage where an additional 25 patients will be 
enrolled. If there are at least 4 responses among the 37 evaluable patients, the regimen would be 
considered to be of clinical benefit worthy of further study (exact alpha=0.09). If the true the 
response rate is 5%, the probability that the study would discontinue after stage I is 54%. The 
total accrual to the study will be 38 given the additional patient added for evaluation in the first 
stage.  
13.3
Analysis of Primary Endpoints
Best overall response will be assessed among patients who received at least one dose of protocol 
therapy using RECIST 1.1 criteria.  The objective response rate (CR+PR) will be reported with 
95% confidence interval adjusting for two-stage design.
13.4
Analysis of Secondary Endpoints
Overall Survival: Overall Survival (OS) is defined as the time from randomization (or 
registration) to death due to any cause, or censored at date last known alive.
Progression-Free Survival: Progression-Free Survival (PFS) is defined as the time from 
randomization (or registration) to the earlier of progression (per RECIST 1.1) or death 
due to any cause. Participants alive without disease progression are censored at date of 
last disease evaluation.
PFS and OS will be described using the methods of Kaplan-Meier, and they will be 
presented with 95% confidence intervals.
Disease control rate: Disease control is defined as CR, PR, or SD ≥ 16weeks per 
RECIST 1.1. Disease control rate will be reported with a 95% exact confidence interval.  
Clinical benefit rate: is defined as CR, PR, or SD ≥ 24weeks per RECIST 1.1. Clinical 
benefit rate will be reported with a 95% exact confidence interval.  
Phospho-Rb reduction will be evaluated as a dichotomous endpoint from paired tumor 
assessment. It is anticipated that 60-70% of patients will have reduction in phospho-Rb 
after one cycle of protocol treatment.  Due to the low anticipated response rate, disease 
control will be used as the primary evaluation of the marker in order to maintain power to 
detect an association with phospho-Rb reduction. Based on data from the Monarch-1 
study in ER+ breast cancer (Dickler et al, ASCO 2016) a 34% disease control rate in the 
total study population would be anticipated if abemaciclib monotherapy is observed to 
have activity (lower than that observed in ER+ disease).
Assuming that 33 pairs of biopsies were evaluable for phosphor-Rb (10% failure rate), 
we will have more than 85% power to detect a difference in the disease control rate if the 
rates are 53.3% among patients with phospho-Rb reduction and 5% among patients 
without phospho-Rb reduction. The following table gives the power of detecting the 
difference in disease control among patients with and without phospho-Rb reduction, 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
54
assuming the overall rate is 34% and with incidences of 60% and 70%  phospho-Rb 
reduction
# of 
paired 
biopsies 
evaluable 
for 
phosphor-
Rb
Expected 
tumor with 
phospho-Rb 
reduction
DCR among 
pts with 
phospho-Rb 
reduction
DCR among 
pts without 
phospho-Rb 
reduction
Power
53.3%
5%
81%
60%
50%
10%
56%
47.7%
2%
73%
29
70%
46.4%
5%
58%
53.3%
5%
85%
60%
50%
10%
63%
47.7%
2%
76%
33
70%
46.4%
5%
60%
.
Phospho-Rb reduction will be evaluated using Fisher Exact test with two-sided alpha = 
0.05. Exploratory analyses will additionally consider the other efficacy endpoints and 
quantitative measures of phospho-Rb reduction using logistic regression models.  With a 
20% probability of objective response overall, there will be 89% power to detect an 
improvement to 50% probability of response with a 1 standardized-unit increase in 
phosph-RB reduction (OR = 4.0). Power calculations were performed in East v6.3 (Cytel 
Inc.).  
13.5
Analysis of Exploratory Endpoints
Descriptive statistics will be used to characterize any types of biomarkers discovered in 
correlative work, T-cell phenotypes via serial PBMCs blood draws (prior to receiving 
therapy, while on therapy, and at the time of progression), any changes in the serial 
microbiome collection, and its correlation with participant outcomes. 
13.6
Reporting and Exclusions
13.6.1 Evaluation of toxicity
All participants will be evaluable for toxicity from the time of their first treatment.
13.6.2 Evaluation of the Primary Efficacy Endpoint
All Participants who receive at least one dose of either study drug will be evaluable for 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
55
efficacy endpoints.  Participants who never start protocol therapy will be considered 
inevaluable and will be replaced.  Biomarker analyses will be conducted in all Participants 
with evaluable biospecimen.
14. PUBLICATION PLAN
The results should be made public within 24 months of reaching the end of the study. The end of 
the study is the time point at which the last data items are to be reported, or after the outcome 
data are sufficiently mature for analysis, as defined in the section on sample size, accrual rate and 
study duration. If a report is planned to be published in a peer-reviewed journal, then that initial 
release may be an abstract that meets the requirements of the International Committee of Medical 
Journal Editors. A full report of the outcomes should be made public no later than three (3) years 
after the end of the study. 
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
56
15. REFERENCES
1.
Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2013. CA Cancer J Clin, 
2013. 63(1): p. 11-30.
2.
Carey, L., et al., Triple-negative breast cancer: disease entity or title of convenience? Nat 
Rev Clin Oncol, 2010. 7(12): p. 683-92.
3.
Anders, C. and L.A. Carey, Understanding and treating triple-negative breast cancer. 
Oncology (Williston Park), 2008. 22(11): p. 1233-9; discussion 1239-40, 1243.
4.
Carey, L.A., et al., The triple negative paradox: primary tumor chemosensitivity of breast 
cancer subtypes. Clin Cancer Res, 2007. 13(8): p. 2329-34.
5.
Finn, R.S., et al., The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination 
with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, 
HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 
study. Lancet Oncol, 2015. 16(1): p. 25-35.
6.
Witkiewicz, A.K. and E.S. Knudsen, Retinoblastoma tumor suppressor pathway in breast 
cancer: prognosis, precision medicine, and therapeutic interventions. Breast Cancer Res, 
2014. 16(3): p. 207.
7.
Lehmann, B.D., J.A. Pietenpol, and A.R. Tan, Triple-negative breast cancer: molecular 
subtypes and new targets for therapy. Am Soc Clin Oncol Educ Book, 2015: p. e31-9.
8.
Lehmann, B.D., et al., Identification of human triple-negative breast cancer subtypes and 
preclinical models for selection of targeted therapies. J Clin Invest, 2011. 121(7): p. 
2750-67.
9.
T.A., T. and M. Kathy, Results from a phase 2 study of enzalutamide (ENZA), an 
androgen receptor (AR) inhibitor, in advanced AR+ triple-negative breast cancer 
(TNBC), in 2015 ASCO Annual Meeting Abstract. 2015.
10.
Asghar, U., et al., The history and future of targeting cyclin-dependent kinases in cancer 
therapy. Nat Rev Drug Discov, 2015. 14(2): p. 130-46.
11.
Trere, D., et al., High prevalence of retinoblastoma protein loss in triple-negative breast 
cancers and its association with a good prognosis in patients treated with adjuvant 
chemotherapy. Ann Oncol, 2009. 20(11): p. 1818-23.
12.
Stefansson, O.A., et al., CpG island hypermethylation of BRCA1 and loss of pRb as co-
occurring events in basal/triple-negative breast cancer. Epigenetics, 2011. 6(5): p. 638-
49.
13.
Herschkowitz, J.I., et al., The functional loss of the retinoblastoma tumour suppressor is 
a common event in basal-like and luminal B breast carcinomas. Breast Cancer Res, 2008. 
10(5): p. R75.
14.
Dean, J.L., et al., Therapeutic response to CDK4/6 inhibition in breast cancer defined by 
ex vivo analyses of human tumors. Cell Cycle, 2012. 11(14): p. 2756-61.
15.
Sherr, C.J., Cancer cell cycles. Science, 1996. 274(5293): p. 1672-7.
16.
Ortega, C., et al., On-line complexation/cloud point preconcentration for the sensitive 
determination of dysprosium in urine by flow injection inductively coupled plasma-
optical emission spectrometry. (1618-2642 (Print)).
17.
Rizzo, J.D., et al., Use of epoetin and darbepoetin in patients with cancer: 2007 
American Society of Clinical Oncology/American Society of Hematology clinical practice 
guideline update. (1527-7755 (Electronic)).
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
57
18.
Parker, J.S., et al., Supervised risk predictor of breast cancer based on intrinsic subtypes. 
J Clin Oncol, 2009. 27(8): p. 1160-7.
19.
Burstein, M.D., et al., Comprehensive genomic analysis identifies novel subtypes and 
targets of triple-negative breast cancer. Clin Cancer Res, 2015. 21(7): p. 1688-98.
20.
Gong, X., et al., Genomic aberrations that activate D-type cyclins are associated with 
enhanced sensitivity to the CDK4 and CDK6 inhibitor abemaciclib. Cancer Cell, 2017. 
32(6): p. 761-776.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
58
APPENDIX A:PERFORMANCE STATUS CRITERIA
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Description
100
Normal, no complaints, no evidence 
of disease.
0
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction.
90
Able to carry on normal activity; 
minor signs or symptoms of disease.
80
Normal activity with effort; some 
signs or symptoms of disease.
1
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work).
70
Cares for self, unable to carry on 
normal activity or to do active work.
60
Requires occasional assistance, but 
is able to care for most of his/her 
needs.
2
In bed <50% of the time.  
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities.  Up and about 
more than 50% of waking hours.
50
Requires considerable assistance and 
frequent medical care.
40
Disabled, requires special care and 
assistance.
3
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours.
30
Severely disabled, hospitalization 
indicated.  Death not imminent.
20
Very sick, hospitalization indicated. 
Death not imminent.
4
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair.
10
Moribund, fatal processes 
progressing rapidly.
5
Dead.
0
Dead.
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
59
APPENDIX B: USE OF STRECK TUBES
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
60
APPENDIX C: INDUCERS AND STRONG INHIBITORS OF CYP3A4 OR 
SUBSTRATES OF CYPS WITH NARROW THERAPEUTIC RANGE
Inducers of CYP3A4
Carbamazepine 
Dexamethasone
Phenobarbital/phenobarbitone 
Phenytoin 
Rifapentine 
Rifampin 
Rifabutin 
St. John’s wort 
Cytochrome P450 Substrates with Narrow 
Therapeutic Range
CYP1A2
Theophylline
Tizanidine
CYP2C8
Paclitaxel
Strong Inhibitors of CYP3A4
Nefazodone
All HIV protease inhibitors
Clarithromycin 
Itraconazole 
Ketoconazole
CYP2C9
Warfarin
Phenytoin 
CYP2D6
Thioridazine
Pimozide
 
 
 
 
 
 


DF/HCC Protocol #:  17-024
Protocol Version Date: 11/11/2019
61
APPENDIX D
GENERATION OF PBMCS
1. Pour blood from green-cap tubes (heparin treated tubes) into two 50 ml conical tubes 
(Corning, 430290).
2. Spin tubes at 1500 rpm for 10 min (Sorvall Legend XTR centrifuge).
3. Aspirate 2 ml plasma/tube and aliquot into 4 tubes mircocentrifuge tubes (Fisherbrand, 
05-408-138) 
4. Spin plasma at 3000 RPM for 5 minutes (Sorvall Legend Micro 21R centrifuge)
5. Aspirate plasma into Cryogenic tubes 2 ml plasma/ tube (Corning, 430488).
6. Dilute blood 1:1 with PBS. (Blood amount should not exceed 25 ml per tube.)
7. Take 2 new 50 ml conical tubes and add 12 ml ficoll-paque (Cat# 17144003; GE 
Healthcare) per tube.
8. Slowly and gently layer the diluted blood on top of the ficoll-paque of the tube with a 
maximum volume of 35 ml.
9. Centrifuge the tube at 1900 rpm for 20 min at room temperature with slow acceleration 
(#7) and deceleration (#7) (Sorvall Legend XTR centrifuge).
10. Remove the PBMC layer from between the upper layer (diluted plasma) and middle layer 
(ficoll-paque) and transfer into a 50 ml conical tube. The lower layer is composed of red 
blood cells.
11. Completely fill conical tube containing isolated PBMC with PBS, mixing well.
12. Count viable cells by mixing 10 µl Trypan Blue with 10µl PBMC/PBS dilution in a 
microcentrifuge tube. Load 10 µl of mixture onto Countess Cell Counting Chamber Slide 
(Invitrogen, C10283) and read with Countess Automated Cell Counter (Invitrogen). 
13. Centrifuge the tubes containing PBMC/PBS mixture at 1500 rpm for 5 min at room 
temperature (Sorvall Legend XTR centrifuge). 
14. Remove PBS, and resuspend PBMC pellet in appropriate amount of freezing solution so 
that there are approx 5 × 106 cells/cryo vial in 300-500 µl of Fetal Bovine Serum (heat 
inactivated) plus 15% DMSO.   
15. Put vials in CoolCell container (Biocision Inc.) and transfer to -80C freezer overnight. 
16. Transfer cells to liquid nitrogen tank