Conditions: Adult Solid Tumor Adenocarcinoma of the Colon Adenocarcinoma of the Rectum Adult Central Nervous System Germ Cell Tumor Adult Teratoma Benign Teratoma Estrogen Receptor-negative Breast Cancer Estrogen Receptor-positive Breast Cancer Familial Testicular Germ Cell Tumor HER2-negative Breast Cancer HER2-positive Breast Cancer Male Breast Cancer Ovarian Immature Teratoma Ovarian Mature Teratoma Ovarian Monodermal and Highly Specialized Teratoma Progesterone Receptor-negative Breast Cancer Progesterone Receptor-positive Breast Cancer Recurrent Breast Cancer Recurrent Colon Cancer Recurrent Extragonadal Germ Cell Tumor Recurrent Extragonadal Non-seminomatous Germ Cell Tumor Recurrent Extragonadal Seminoma Recurrent Malignant Testicular Germ Cell Tumor Recurrent Melanoma Recurrent Ovarian Germ Cell Tumor Recurrent Rectal Cancer Stage III Extragonadal Non-seminomatous Germ Cell Tumor Stage III Extragonadal Seminoma Stage III Malignant Testicular Germ Cell Tumor Stage III Ovarian Germ Cell Tumor Stage IV Breast Cancer Stage IV Colon Cancer Stage IV Extragonadal Non-seminomatous Germ Cell Tumor Stage IV Extragonadal Seminoma Stage IV Melanoma Stage IV Ovarian Germ Cell Tumor Stage IV Rectal Cancer Testicular Immature Teratoma Testicular Mature Teratoma
Allocation: NON_RANDOMIZED | Intervention model: SINGLE_GROUP
Enrollment: 304
Healthy volunteers: no

Brief summary:
RATIONALE: PD 0332991 may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth.

PURPOSE: This phase II trial is studying the side effects and how well PD 0332991 works in treating patients with refractory solid tumors.

Detailed description:
PRIMARY OBJECTIVES:

I. To determine the response rates following treatment with PD 0332991 in the following malignancies: 1) Metastatic breast cancer, 2) Metastatic colorectal cancer, 3) Metastatic melanoma with CDK4 mutation or amplification, or 4) Cisplatin-refractory, unresectable germ cell tumors.

OUTLINE:

Patients receive oral PD 0332991 once daily on days 1-21. Treatment repeats every 28 days for up to 12 courses in the absence of unacceptable toxicity or disease progression.

Arms:
arm 1: Metastatic breast cancer

PD-0332991 Given orally, 125 mg QD on a 21-day arm 2: Metastatic colorectal cancer that harbors the Kras or BRAF mutation

PD-0332991 Given orally, 125 mg QD on a 21-day arm 3: Advanced or metastatic esophageal and/or gastric cancer

PD-0332991 Given orally, 125 mg QD on a 21-day arm 4: Cisplatin-refractory, unresectable germ cell tumors

PD-0332991 Given orally, 125 mg QD on a 21-day arm 5: Any tumor type if tissue tests positive for CCND1 amplification, CDK4/6 mutation , CCND2 amplification OR any other functional alteration at the G1/S checkpoint.

PD-0332991 Given orally, 125 mg QD on a 21-day

Interventions: intervention 1: PD-0332991
Intervention descriptions: intervention 1: Given orally, 125 mg QD on a 21-day

Protocol document:
Prot_000.pdf: 
 
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1
PHASE II TRIAL OF THE CYCLIN-DEPEDENT KINASE INHIBITOR PD 0332991 IN PATIENTS 
WITH CANCER 
 
 
 
Principal Investigator:  Peter J. O’Dwyer, MD 
 
Co-Investigators:   
 
Ravi Amaravadi 
David Vaughn 
Ursina Teitelbaum 
Angela DeMichele 
Susan Domchek 
Amy Clark 
Kevin Fox 
Michael Kochman 
Gregory Ginsberg 
Daniel Pryma 
David Mankoff 
Pallavi Kumar 
Naomi Haas 
                                                                         Vivek Narayan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Compound: PD 0332991  
UPCC#03909   
Phase: 1-2  
Version dated: 04/18/2018 


 
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SYNOPSIS 
TITLE  
Phase II trial of the cyclin-dependent kinase inhibitor PD 0332991 in 
patients with cancer 
PROTOCOL NUMBER 
PD 0332991 
 
CLINICAL PHASE 
Phase II 
 
RATIONALE 
PD 0332991 is a novel, potent and selective inhibitor of cyclin-dependent 
kinases 4 & 6. PD 0332991 causes cell cycle arrest and induces apoptosis 
in cells containing detectable levels of the phosphorylated form of the 
retinoblastoma protein (Rb+).  Dysregulation of the cell cycle is a 
hallmark of cancer, and frequently involves cdk4 and 6, or cyclin D, their 
principal cyclin partner.   
 
Tumor regression was observed in patients treated in Phase I trials of PD 
0332991 at doses that were tolerable.  The major toxicity was neutropenia, 
which was mild to moderate at the dose to be utilized in this Phase II trial.  
At these doses in a separate proof-of-concept study, evidence of inhibition 
of the cell cycle was demonstrated in imaging of tumor uptake of 
thymidine by FLT-PET.  In this study, the activity of  PD 0332991 will be 
sought in patients with a group of cancer types which for various reasons 
may be enriched for potential responders, and in whom detailed studies to 
elucidate PK/PD determinants of drug effect and correlative biological 
analyses will be performed.  
OBJECTIVES 
 
The primary objectives of this study are: 
• To determine the response rates following treatment with PD 
0332991 in the following malignancies: 
1) Metastatic breast cancer  
 
2) Metastatic colorectal cancer that harbors the Kras or BRAF 
mutation  
3)  Advanced or metastatic esophageal and/or gastric cancer 
 
4) Cisplatin-refractory, unresectable germ cell tumors 
 
5) Any tumor type if tissue tests positive for CCND1 amplification, 
CDK4/6 mutation, CCND2 amplification OR any other functional 
alteration of the G1/S checkpoint. 
 
• To evaluate the safety and tolerability of PD 0332991, 
administered to subjects with refractory solid tumors 
The secondary objectives of this study are: 
• 
To assess the pharmacodynamic effects of PD 0332991 on tumor 
and non-tumor tissue  
• 
To investigate the relationship between selected biomarkers, PK 
and/or efficacy and safety outcomes. 
 


 
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• 
To estimate the population pharmacokinetics for PD 0332991 and 
to correlate PK with efficacy outcomes. 
• 
 
• 
To evaluate the preliminary safety and efficacy of PD 0332991 in 
combination with trastusumab in patients with HER2-
overexpressing tumors within the 5 cohorts under study 
• 
To perform a Phase II evaluation of PD0332991 in a population 
defined as potential responders on the basis of CCND1 gene 
amplification. 
 
 


 
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STUDY DESIGN 
This is a phase II, non-randomized, open-label study, evaluating the 
response rates, time to progression, safety, tolerability, PK and 
pharmacodynamics of PD 0332991 in subjects with selected solid tumors. 
Patients will be treated with the drug at a dose of 125 mg QD on a 21-day 
schedule, with cycles repeating every 28 days. 
 
The study will consist of the following periods: 
Pre-Treatment Period: Subjects are consented and qualified for the study. 
Treatment Period: Subjects will be treated and monitored for safety 
(including laboratory assessments) and signs of toxicity.  They will be 
evaluated every two to three cycles by conventional imaging modalities to 
determine response (measured using RECIST criteria).   Imaging and 
biopsy of accessible disease pre-treatment and during the first cycle of 
therapy will be used to assess pharmacodynamic endpoints.  In the 
absence of progressive disease (PD) and unacceptable PD 
0332991-related toxicity, subjects may continue to receive drug treatment 
until progression. 
Post-Treatment Period:    Additional follow-up will occur for significant 
adverse events related to study treatment that are not resolved by or occur 
after the time of the last visit. 
 
NUMBER OF SUBJECTS 
Approximately 100-205 subjects will be treated in 5 cohorts  
 
TARGET POPULATION:  
INCLUSION CRITERIA: 
1. Disease Characteristics: 
All Subjects:  All subjects treated under this protocol will have 
histologically documented cancer of one of the following types:  
 
A.  Metastatic breast cancer (7 triple negative, 23 ER+ after the first 15 
patients are enrolled on the non-CCND1 cohort; in addition 10 HER2+ for 
combination trastuzumab and PD0332991 therapy) up to 55 total 
enrollment slots  
 
B.  Metastatic colorectal cancer that harbors the Kras or BRAF mutation 
(15-30 enrollment slots) 
 
 C.  Advanced or metastatic esophageal and/or gastric cancer (15-30 
enrollment slots) 
 
D.   Cisplatin-refractory, unresectable germ cell tumors (15-30 enrollment 
slots) 
 
E.  Any tumor type if tissue tests positive for CCND1 amplification, 
CDK4/6 mutation , CCND2 amplification OR any other functional 
alteration at the G1/S checkpoint. (15-30 enrollment slots) 
 
 
2. Biopsy Requirements:   
For Subjects with accessible disease amenable to biopsy: 


 
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A biopsy will be obtained pre-treatment and during Cycle 1 (while 
patient is receiving drug) for molecular markers of the cell cycle, and 
its inhibition.  
 
For all subjects: 
1. Subjects will be > 18 years old 
2. The subject has disease that is assessable by tumor marker, physical, 
or radiologic means. 
3. The subject has an Eastern Cooperative Oncology Group (ECOG) 
performance status of 0 or 1. 
4. The subject has adequate organ function, defined as follows 
a. Bilirubin ≤ 1.5 x the upper limit of normal (ULN) 
b. Serum creatinine ≤ 1.5 x UNL or calculated creatinine clearance ≥ 
60 mL/min, and  
c.   For subjects without liver metastases: alanine aminotransferase 
(ALT) and aspartate aminotransferase (AST) ≤ 2.5 x ULN 
d. For subjects with liver metastases: alanine aminotransferase 
(ALT) and aspartate aminotransferase ≤ 5 x ULN   
 5.    All tumors must test positive for Rb expression with the 
exception of:       
 a. ER positive metastatic breast tumors (data now shows all to 
be Rb positive.) 
       b. Any tumor type if tissue tests positive for CCND1 
amplification, CDK4/6 mutation , CCND2 amplification OR 
any other functional alteration at the G1/S checkpoint. 
6. The subject has adequate marrow function, defined as follows 
a. Absolute neutrophil count (ANC) >1500/mm3 
b. Platelets >100,000/mm3, and 
c.  Hemoglobin > 9 g/dL 
7. The subject is capable of understanding and complying with the 
protocol requirements and has signed the informed consent 
document. 
8. Sexually active subjects (male and female) must use accepted 
methods of contraception during the course of the study and for 
3 months after the last dose of protocol drug(s). 
9. Female subjects of childbearing potential must have a negative 
pregnancy test at screening. Females of childbearing potential are 
defined as sexually mature women without prior hysterectomy or 
who have had any evidence of menses in the past 12 months. 
10. However, women who have been amenorrheic for 12 or more months 
are still considered to be of childbearing potential if the amenorrhea is 
possibly due to prior chemotherapy, antiestrogens, or ovarian 
suppression. 
 
EXCLUSION CRITERIA: 
1. The subject has received cytotoxic chemotherapy (including 
investigational cytotoxic chemotherapy) within 3 weeks (or 
nitrosoureas or mitomycin C within 6 weeks) before the first dose of 
PD 0332991.  Patients with HER2-overexpressing tumors may receive 


 
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trastuzumab up to the date of starting therapy, and may continue to 
receive trastuzumab while receiving PD0332991. 
2. The subject has received any other type of investigational agent within 
28 days before the first dose of study treatment.  
3. The subject has not recovered from clinically-meaningful toxicity due 
to prior therapy (i.e., back to baseline or Grade ≤ 1) with the exception 
of neurotoxicity and alopecia. 
4. The subject has untreated or uncontrolled brain metastases or 
evidence of leptomeningeal involvement of disease unless the 
subject has a teratoma in which case s/he may be eligible if all 
other eligibility criteria are met 
5. The subject has uncontrolled intercurrent illness including, but not 
limited to: 
a. ongoing or active infection 
b. diabetes mellitus 
c. hypertension 
d. symptomatic congestive heart failure, unstable angina pectoris, 
stroke or myocardial infarction within 3 months 
6.  The subject has a baseline corrected QT interval (QTc)  
> 470 ms.  
 
7.  The subject is pregnant or breastfeeding. 
 
8.  The subject is known to be positive for the human immunodeficiency 
virus (HIV). Note: baseline HIV screening is not required 
 
9.  The subject is unable or unwilling to abide by the study protocol or 
cooperate fully with the investigator or designee. 
 
ESTIMATED LENGTH 
OF STUDY 
Approximately 10 years for subject accrual and treatment. 
ESTIMATED STUDY 
DATES 
October 2009 (first patient, first visit) to September, 2019 (last patient, 
first visit) 
INVESTIGATIONAL 
REGIMEN 
DOSE/ROUTE/ 
DURATION 
Cycles will be 28 days in duration. For all subjects, PD 0332991 will be 
administered at a dose of 125mg daily as a single dose for 21 days out of 
28, cycles defined as 28 days.  Patients with HER2-overexpressing tumors 
have the option of receiving trastuzumab, at standard dose, scheduled 
every three weeks. 
COMPARATOR DRUG 
None 
 
ASSESSMENTS: 
 
SAFETY 
Safety will be assessed by evaluation of adverse events, vital signs, ECG, 
laboratory tests and concomitant medications. Adverse event seriousness, 
severity grade and relationship to study treatment will be assessed by the 
Investigator. Severity grade will be defined by the National Cancer 


 
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Institute CTCAE v5.0.  For patients receiving concurrent trastuzumab, 
cardiac evaluation with either echocardiogram or MUGA scan will be 
obtained prior to starting PD0332991 and every 12 weeks (+/- 1 week) 
while receiving PD0332991. 
TUMOR ASSESSMENTS 
Tumor response for subjects with measurable lesions will be assessed 
using the Response Evaluation Criteria in Solid Tumors (RECIST) 
criteria. Subjects with measurable lesions should be assessed using 
computerized tomography (CT) or magnetic resonance imaging (MRI) 
scan at baseline and approximately every 9 weeks (± 4 days) from date of 
first dose of PD 0332991 until documented progression. 
 
PHARMACOKINETICS 
Blood samples will be obtained at limited time points, and matched with 
tumor biopsy, biomarker and hematology assessments. 
 
PHARMACODYNAMICS 
For all patients, blood specimens for analysis of PD0332991 will be 
collected: (1) On Cycle 1 Day 14 at pre-dose; (2) On Cycle 1 Day 15 at 
pre-dose, at 4 hours post-dose and between 7-10 hours post-dose, (2) 
matched with FLT-PET assessments except at baseline, (3) matched with 
tumor assessments except at baseline.  A number of patients with 
accessible tumor will have biopsies, and have the tumor biopsy divided, 
with one aliquot flash frozen, and the other processed for FFPE.  Samples 
to be analyzed for markers of cell cycle inhibition (such as Ki67, and 
others) and emerging markers of sensitivity to cell cycler inhibition.  
STATISTICAL METHODS The response rate of interest in this trial will be 15% for each of the 
indications tested.   If we observe at least one response in the first fifteen 
subjects, we will enroll a further 15 subjects, for a total of 30.  Observing 
zero responses in the first 15 subjects would exclude response rates as low 
as 15% with a 90% one-sided upper confidence bound. 
 
If at least three responses are observed in a total of 30 evaluable patients, 
we will conclude that the drug is active and merits further study; if the true 
response rate is 15% or greater, three or more responses will be observed 
with a probability of at least 85%.  If the true response rate is only 3%, 
three or more responses will be observed with a probability of only 6%.  In 
addition to responses of CRs and PRs, disease stabilization will be 
considered: if the proportion of patients with disease stabilization for 6 
months exceeds 20%, we will conclude that the drug may warrant further 
investigation in that indication.  
 
Confidence intervals will be calculated for selected safety and exploratory 
variables. Adverse event terms will be standardized using the Medical 
Dictionary for Regulatory Activities (MedDRA) and tabulated by system 
organ class and preferred term. Laboratory parameters will be 
summarized using mean change in value and/or shift in category since 
baseline. 
 
Pharmacokinetic data from this study may be analyzed using mixed-effect 


 
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modeling approaches.  The intent of this analysis will be to build upon a 
basic population pharmacokinetic model for PD 0332991 and to 
determine the inter-individual and residual variability in population PK 
parameters of interest. If data permit, PK/PD correlations to investigate 
any causal relationship between PD 0332991 exposure and efficacy 
outcomes will be explored. 
SUV calculations will be used to assess PET responses.  By convention a 
positive PET response will be taken as a decrease in summed SUV values 
of > 25%.  Relationships will be sought between PET responses and CT 
imaging response, as well as with PK indices in individual patients. 
We will target a total accrual (for treatment) of 100-205 patients, 
depending on the availability of continued funding (the screening is 
projected to be the most resource-intensive part of this proposal, and our 
budget assumptions are based on a rate of 15% positivity – we will need 
to screen about 350 patients.  A response rate of interest will be 20% 
(though clearly a high proportion of patients with stable disease could be 
considered interesting in some diseases).  Taking that as a cut-off, we will 
treat patients with any one of these diseases until 15 have been accrued.  
If there have been no responses we will conclude with 95% confidence 
that the true response rate is less than 20% and no further patients will be 
screened.  If one or more responses are observed, we will continue accrual 
in that disease type to a total of 30.  For the population overall, a response 
rate of 15% or more, or prolonged (> 6 months) stable disease in 30% or 
more, would be convincing evidence of drug action in a targeted 
population, and would prompt additional studies. 
 
 
 
 
 


 
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TABLE OF CONTENTS 
1.0 BACKGROUND AND RATIONALE.......................................................................11 
 
2.0 STUDY OBJECTIVES AND ENDPOINTS..............................................................20 
 
3.0 STUDY DESIGN........................................................................................................21 
 
4.0 ELIGIBILITY………………….................................................................................21 
 
5.0 STUDY TREATMENTS.............................................................................................24 
 
6.0 STUDY PROCEDURES ............................................................................................28 
 
7.0  ASSESSMENTS.........................................................................................................29 
 
8.0  ADVERSE EVENT REPORTING............................................................................34 
 
9.0  DATA ANALYSIS/STATISTICAL METHODS......................................................37 
 
10.0. REFERENCES ..........................................................................................................39 
 
 


 
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List of Abbreviations 
AE 
adverse event 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
AR 
accumulation ratio 
AST 
aspartate aminotransferase 
AUC 
area under the plasma concentration time curve 
AUC0-∞ 
area under the plasma concentration time curve from time of dosing to infinity 
AUC0-τ 
area under the plasma concentration time curve from time of dosing to τ, where τ is 
equal to dosing interval 
BUN 
blood urea nitrogen 
CFR 
code of federal regulations 
Cmax 
maximum plasma concentration 
Cmin 
minimum plasma concentration  
CL 
plasma clearance after IV administration 
CL/F 
apparent plasma clearance after PO administration 
CR 
complete response 
CRC 
cohort review committee 
CRO 
contract research organization 
CRF 
case report form 
CNS 
central nervous system 
CT 
computerized tomography 
CTCAE 
common terminology criteria for adverse events 
CYP 
cytochrome p450 
DLT 
dose-limiting toxicity 
DSMC 
data safety monitoring committee 
EC 
ethics committee 
ECG 
electrocardiogram 
ECOG 
eastern cooperative oncology group  
ED50 
dose required for 50% tumor inhibition 
F 
absolute bioavailability after PO administration 
FDA 
food and drug administration 
FLT 
fms-like tyrosine kinase 
GCP 
good clinical practice 
HR 
hazard ratio 
ICH 
international conference on harmonization 
ICON 4 
international collaborative ovarian neoplasm 4 
INR 
international normalized ratio 


 
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List of Abbreviations 
IRB 
institutional review board 
KDR 
kinase insert domain receptor 
MedDRA 
medical dictionary for regulatory activities 
MRI 
magnetic resonance imaging 
MAD 
maximum administered dose 
MTD 
maximum tolerated dose 
NOAEL 
no observable adverse effect level 
PD 
Progressive disease 
PFS 
progression free survival 
PK 
pharmacokinetic 
PR 
partial response 
PT/PTT 
prothrombin time/partial thromboplastin time 
RBC 
red blood cell 
RECIST 
response evaluation criteria in solid tumors 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SD 
stable disease 
t1/2 
terminal half-life 
tmax 
observed time to reach peak plasma concentration 
UNL 
upper normal limit 
WBC 
white blood cell 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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1.0 
BACKGROUND AND RATIONALE 
 
1.1  Cell Cycle Dysregulation and Cancer 
 
One of the hallmarks of cancer is cell cycle dysregulation, either through overactivity of the proliferative 
machinery (as by activating mutations in cyclin/cdk components, gene amplification of regions of the 
genome that encode them, or overexpression of individual proteins that relate to cell cycle proteins) or 
through inactivation of the proteins that serve to block the cell cycle when appropriate (such as 
inactivating mutations in cell cycle inhibitors such as p53 or p16, or of components of the Rb complex), 
with the consequence that cells adopt a hyperproliferative program (1).  A key regulator of the cell cycle, 
Rb activity is decreased or lost in a variable proportion of common epithelial malignancies (2).  The Rb 
protein is a transcriptional repressor of the cell cycle, which in its unstimulated form binds the promoter 
region of E2F to shut down the transcription of genes required for DNA replication and mitosis.  
Phosphorylation of Rb (as initiated by cdk’s 4 & 6 in complexes with cyclin D) displaces the protein from 
the E2F promoter, and permits the progression of the cell cycle through mitosis, when the action of 
phosphatases restores the hypophosphorylated form and again represses the E2F transcriptional program.  
The early association of Rb deficiency and retinoblastoma established the importance of the integrity of 
this pathway in protection from developing cancer, and led to a focus on the cell cycle as a target for 
cancer control (3-8).  Work in recent years has elucidated the multiple interactions of Rb and other cell 
cycle regulators with cellular processes in addition to proliferation, including apoptosis, senescence, and 
genomic stability (9).  It has become clear that inactivating lesions in many of these proteins, including 
Rb, may have consequences for cell survival that are cell type- and context-dependent.  Therefore studies 
of agents designed to impact cell cycle regulatory pathways will be most informative if they assess drug 
effects at multiple levels, and establish in as detailed a manner possible the context in which the treatment 
is administered (ie, the molecular characteristics of the tumor).   
 
 
1.2  Cell Cycle Abnormalities in specific cancers 
 
Abnormalities of cell cycle regulation are characteristic of most solid tumors (2,9).  Inactivation of p53 is 
believed to be a characteristic of some 50% of cancers, and the result is loss of the cdk inhibitor p21.  In 
40 – 60% of tumors p16 function is lost, and in a smaller proportion Rb function is compromised or lost.  
Interestingly, despite what is therefore a ubiquitous characteristic of the cancer cell, it is usually difficult 
to establish a clear relationship to prognosis for any of these abnormalities, and this perhaps serves to 
further emphasize the importance of context (accompanying molecular abnormalities) in determining the 
malignant behavior of these tumors.  There are several examples of cancers in which dysregulation of 
specific components of the cell cycle can be shown to drive proliferation.  With respect to the cyclins and 
cyclin-dependent kinases, overexpression of cyclin D1 is observed in several tumor types.  This is most 
commonly achieved by gene amplification or transcriptional mechanisms, but in the case of mantle cell 
lymphoma, a translocation is responsible.   
 
 
 
1.2.1  CDK inhibition in Breast Cancer 
 
Overexpression of canonical cyclin D1a is common  in breast carcinoma, with up to 50% of primary 
ductal breast carcinomas overexpressing cyclin D1a in various studies (10,11).    Increased expression has 
been shown to be evident in all histological types of breast cancer and is present in both estrogen receptor 
positive and estrogen receptor negative malignancies (12,13).  Once acquired, cyclin D1a overexpression 
is maintained through all stages of disease, including metastatic lesions (14).  Despite the undisputed 


 
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presence of cyclin D1a in breast cancer, data thus far have produced conflicting results regarding the 
biological and clinical significance of cyclin D1a overexpression. Cyclin D1a overexpression alone has 
not been shown to induce malignant transformation in cell lines and mouse models, but nuclear cyclin D1 
alleles have exhibited increased oncogenic potential. (15). 
 
A polymorphism in the CCND1 gene, G870A, results in an aberrantly spliced protein (“cyclin D1b”) 
lacking the Thr-286 phosphorylation site necessary for nuclear export.   Studies of murine fibroblasts have 
shown that although overexpression of canonical cyclin D1 (“cyclin D1a”) alone is not sufficient to drive 
malignant transformation, expression of nuclear cyclin D1b is oncogenic.  We recently reported the 
results of an analysis of cyclin D1a and D1b splice variant expression in a cohort of 118 patients with 
invasive breast cancer (Gupta, submitted).   Cyclin D1b was expressed in 26% of tumors and cyclin D1a 
was overexpressed in 27%; co-expression occurred in 4%.  Cyclin D1a and/or D1b expression were not 
significantly associated with ER/PR negativity, Her2 overexpression, young age, lymph node positivity, 
high tumor grade, or large tumor size.  With a median follow-up of 44 months, the risk of recurrence was 
higher in those co-expressing D1a and D1b compared to either alone (RR=5.3, 95% CI 1.27 to 22.1, p = 
0.02).  The hazard ratio for those with co-expression compared with those without was 6.05 (p = 0.04).  
Thus, the presence of the splice variant in D1b in addition to overall cyclin D1 overexpression 
characterizes a breast cancer population at high risk of recurrence, who may be particularly suited to CDK 
4/6 inhibition.  The expression of cyclin D1b, and the presence of the mutation that prevents its nuclear 
export, will be assessed retrospectively in breast cancer samples. 
 
 
 
 
1.2.1.1 CDK inhibition in HER2-Overexpressing Breast Cancer 
 
Preclinical and unpublished work by Eric Knudson demonstrated efficacy of PD 0332991 in HER2 
positive breast cancer cell lines as well as in mouse xenograft models (personal communication).  
Furthermore, in tumor models that have failed multiple HER2-directed therapies, PD 0332991 proved to 
be an effective anti-neoplastic (personal communications).  This suggests that the Rb/CDK4/6 pathway 
may be instrumental in resistance to HER2 directed therapy.  Studies in metastatic breast cancer have 
shown that dual HER2-directed targeted therapies have superior efficacy to trastuzumab alone, and that 
the addition of another targeted agent to trastuzumab in the setting of trastuzumab resistance can restore 
sensitivity to this therapy.   However, there are currently no data on the comparative efficacy of 
PD0332991 alone vs. PD0332991 with concurrent trastuzumab.  Toxicities of these agents are non-
overlapping, suggesting an ability to combine these agents safely in patients with HER2-overexpressing 
disease. 
 
 
 
1.2.2  Colon Cancer 
 
Abnormalities of the Rb family (pRB, pRB2/p130, p107) have been identified in a large proportion of 
colon cancers, and are implicated in tumor pathogenesis and progression (16,17). Some colon 
adenocarcinomas undergo allelic loss at the Rb locus (18,19), whereas almost half of colorectal 
carcinomas show nonrandom chromosomal 13 gains (20,21). The loss of Rb and CDK inhibitor 
p16(INK4a) correlates with the poor outcome in colon cancers (22,23). Approximately two thirds of 
intestinal tumors paradoxically have increased levels of Rb expression and protein production when 
compared with paired normal colonic tissue (24), and a mechanism through which Rb may play an 
antiapoptotic role through an interaction with BAG-1 has been proposed (25).  In murine models, APC 
mutant mice engineered to have Rb deletion in the intestine demonstrate accelerated tumor formation 
especially in the large bowel (26).  Thus substantial dysregulation of the Rb-cyclin axis exists in colon 
cancers.  Specific abnormalities of cdk’s and cyclins are also implicated in colon cancer progression.  In 
MSI tumors harboring TGF-β receptor-II mutations, cdk4 expression is markedly increased and may even 


 
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provide a marker for drug effect (27).  More recently, miRNA-mediated cdk6 dysregulation has been 
demonstrated in colon cancer models, but the incidence of this change in clinical samples has not yet been 
reported (28).  Based on these findings, it is appropriate to accrue an unselected patient population in this 
colon cancer trial, with a view to a retrospective biological analysis of responders.   
 
1.2.3  Melanoma 
 
Mutations that underlie activation of the MAP kinase and  PI3 kinase pathways and inactivate the p53/Rb 
pathway are the defining features of melanoma (29-31). The vast majority of melanomas harbor activating 
mutations in c-kit (3%), NRAS (15%), or BRAF (60%). The PI3 kinase pathway is activated through loss 
of PTEN (30%) or amplification of Akt3 (20%) in a large proportion of BRAF mutant melanomas. 
Overlying those alterations, p16 is very commonly mutated or deleted, allowing CDK4 to signal in an 
unopposed fashion. Less commonly cyclin D amplification has been described in melanoma. In cases 
where p16 is intact and cyclin D is not amplified, CDK4 harbors activating mutations allelic amplification 
driving overexpression. We have found that CDK4 mutations mediate resistance to BRAF inhibition, 
when they are present in conjunction with BRAF mutation in melanoma [Figure]. Therefore, CDK4 may 
be a superior target to BRAF in these instances. In our large panel of melanoma cell lines, approximately 
8% harbor activating mutations in CDK4, most commonly the R24C and K22Q substitutions. In addition, 
we have identified a subset of melanomas 
which harbor high level amplification of CDK4 
in conjunction with amplification of c-kit. 
Here, again, we believe that CDK4 inhibition is 
a rational approach. For the past several years, 
we 
have 
been 
prospectively 
evaluating 
patients’ melanomas for the presence of BRAF 
and c-kit mutations for inclusion into clinical 
trials 
with 
relevant 
signal 
transduction 
inhibitors. This screening process can be easily 
modified to include CDK4 mutation or 
amplification as a prospective evaluation. In 
this way, we can identify patients with 
metastatic melanoma, who appear to be clinical 
trial candidates and match them with the most relevant targeted therapy in clinical trials. Enriching for 
patients with CDK4 mutations or amplification will enrich the population for patients with melanoma 
who may be most response to single-agent therapy. If this proof-of-concept is established, it would 
appropriate to further develop the agent for these patients while also exploring the role of the agent in 
combination with other signaling inhibitors in the broader melanoma population as CDK4 signaling 
appears to be aberrant in the vast majority of melanomas through  
 
 
 
 
1.2.4  Germ cell Tumors 
 
Deregulation of the retinoblastoma tumor suppressor (pRB) pathway in germ cell tumors (GCT) has been 
well documented (32).  For example, embryonal carcinomas demonstrate little or no pRB expression.  
However, mature teratomas which are cisplatin- refractory strongly express pRB, suggesting an intact 
G1/S checkpoint.  The extent of pRB expression in other cisplatin-refractory GCT has not been reported. 
Treatment of teratomas and other pRB-positive cisplatin-refractory GCT with CDK 4/6 inhibitor such as 
PD 0332991 is a rational approach.  
 


 
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We previously reported on the clinical course of 3 male patients aged 22 to 37 years with growing 
teratoma syndrome (GTS) treated on a phase I trial of oral daily PD 0332991 in refractory solid tumors 
(NCT00141297) (33).  These patients initially presented with metastatic non-seminomatous GCT.  They 
received cisplatin-based chemotherapy followed by two or more post-chemotherapy surgeries for GTS.  
All developed progressive disease not amenable to further surgery. These teratomas demonstrated strong 
nuclear expression of pRB in the epithelial component.  The first patient received PD 0332991 150 mg 
daily for 21 days on an every 28 day cycle.  The dose was decreased to 100 mg secondary to neutropenia.  
He demonstrated stable disease for 18 months.  The second patient received treatment with PD 0332991 
200 mg daily for 14 days on an every 21 day cycle. The dose was decreased to 150 mg because of 
headaches and vomiting. He also experienced neutropenia.  He has demonstrated a partial response by 
Response Evaluation Criteria in Solid Tumors (RECIST) for 22 months and continues on treatment. The 
third patient was treated with PD 0332991 125 mg daily for 21 days on an every 28 day cycle.  He has 
experienced neutropenia. He has demonstrated stable disease for 24 months and continues on treatment.  
This report is the largest published series of patients with GTS treated with a novel agent and, remarkably, 
all patients derived clinical benefit.  We plan to expand our experience by treating additional patients with 
GTS and other pRB positive cisplatin-refractory GCT with PD 0332991 as part of this phase II study. 
 
1.2.5  Esophageal and/or Gastric Cancer 
 
The role of MAP kinase pathways in mediating multiple cell responses, including proliferation, is well-
recognized (Wagner EF, Nebreda AR.  Signal integration by the JNK and p38 pathways in cancer 
development.  Nature Rev Cancer 9: 537-549, 2009).  A recent publication has focused on signaling 
through the JNK pathway as a mediator of gastric cancer proliferation (Hayakawa Y, Hirata Y, Nakagawa 
H, et al.  Apoptosis signal-regulating kinase 1 and cyclin D1 compose a positive feedback loop 
contributing toi tumor growth in gastric cancer.  Proc Natl Acad Sci USA 108: 780-5, 2011).  Hayakawa 
and colleagues found increased expression of ASK1 (a MAPKKK) in human gastric cancer specimens, 
and in 23/26 gastric cancer cell lines.  Functional relevance oif this finding was suggested by the finding 
of activation of the pathway (elevated p-c-Jun), and cyclin D1, and confirmed through knockdown 
experiments in which ASK1 siRNA inhibited gastric cancer cell growth, while exogenous expression 
induced cell growth through a positive feedback loop involving cyclin D1.  These findings raise the 
possibility that interrupting this growth-stimulatory loop could arrest the growth of gastric cancer in 
humans, a possibility to be tested in a pilot fashion in this trial. 
 
 
1.3  PD 0332991 – an inhibitor of cyclin-dependent kinases 4 & 6 
 
Because increased proliferation is a hallmark of most cancers, agents that specifically inhibit proliferation 
may have a role in the clinical management of cancer. One important target is the cell division cycle, 
which is controlled by the sequential activation of a family of related cyclin-dependent kinases (CDKs). 
CDK4 and CDK6 (CDK4/6) are closely related kinases that promote cell cycle progression from G1 to S 
phase. This is a key regulatory step in the cell division cycle and is followed by commitment to cell 
division (G2 followed by M phase)..  
 
The only known natural substrate for CDK4/6 kinase activity is the retinoblastoma (Rb) protein. In a 
minority of tumors (eg, retinoblastoma, small-cell lung cancer), control of G1/S progression is lost by Rb 
gene mutation. In most common tumors, however, other genetic and epigenetic changes lead to increases 
in CDK4/6 activity and uncontrolled tumor cell growth. Thus, tumor cell proliferation may be particularly 
sensitive to a selective inhibitor of CDK4/6.  
 
The small molecule kinase inhibitor PD 0332991 is a potent inhibitor of both of these cdk’s with in vitro 


 
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inhibitory constants in the nanomolar range (ref). The compound prevents cellular DNA synthesis by 
prohibiting movement from the G1 phase of the cell cycle into the S phase, as demonstrated in laboratory 
models. PD 0332991 preclinical data indicate that it may be expected to have direct cytotoxic activity as 
well as the potential for growth arrest. Arrest in the G1 phase is known to cause cell death in some tumor 
types, as demonstrated by this and other G1 arresting agents (eg, Tamoxifen).  Moreover, CDK inhibitors 
like PD 0332991 have the potential to enhance the effects of conventional therapies.  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.4 
Preclinical Efficacy 
 
Of the 16 phosphorylation sites on Rb, two are known to be specifically phosphorylated by 
CDK4/6;serine-780 and serine-795. The phosphorylation status of Rb at these specific sites in treated 
tumors provides an appropriate biomarker for target modulation that would give assurance that PD 
0332991 is inhibiting its intended target. The IC50 values for reduction of Rb phosphorylation at serine-
780 and serine-795 in MDA-MB-435 breast carcinoma were 0.066 µM and 0.063 µM, respectively. 
Similar effects on serine-780 and serine-795 phosphorylation were obtained in the Colo-205 colon 
carcinoma.  
Time-course experiments indicate that the reduction of Rb phosphorylation begins to occur as quickly as 
four hours after exposure to PD 0332991 and reaches a maximum at 16 hours with continued exposure. 
The inhibition is completely reversible. After removal of the drug, phosphorylation on serine-780 and 
serine-795 begins to return in 2 hours and is complete within 16 hours. Active cellular proliferation 
returns concurrently with Rb phosphorylation. While this suggests that PD 0332991 will be most 
efficacious using continuous daily dosing, intermittent dosing using a variety of schedules has proven as 
efficacious as continuous daily dosing in animal xenograft models. Tumor regrowth is observed following 
discontinuation of therapy. This provides the basis for chronic intermittent dosing, which allows the 
integration of other agents as well as a period for recovery from anticipated toxicities, e.g. 
myelosuppression.  
 
To determine whether tumors that regressed following discontinuation of PD 0332991 remained sensitive 
to further PD 0332991 treatment or whether they had acquired resistance, Colo-205 colon tumors that 
regressed post-PD 0332991 treatment were harvested and re-implanted into naïve mice. Following PD 
0332991 treatment at a dose and schedule identical to the original experiment, the tumors responded with 
equal sensitivity to the drug and fully regressed, indicating that no resistance had developed during the 
initial treatment.  
 
PD 0332991 exhibits significant antitumor efficacy, including tumor regressions, against multiple human 
tumor xenograft models in nude mice. PD 0332991 has produced striking tumor regressions in some 
models and tumor growth delay in others. In the MDA-MB-435 breast carcinoma model, the two dose 
levels that resulted in ≥50% tumor growth inhibition suppressed phosphorylation on serine-780 over the 
full 24-hour prior between doses. At two lower doses, which did not produce ≥50% tumor growth 
inhibition, phosphorylation returned over the 24-hour prior before the next dose. Similar results were 
observed with the Colo-205 colon carcinoma model.  
 
In MDA-MB-435 breast carcinoma cells exposed for 24 hours to varying concentrations of PD 0332991, 
the percentage of cells in G1 began to increase in the presence of as little as 0.04 µM PD 0332991 with a 
concomitant decrease in other phases of the cell cycle. Maximum effects were attained at 0.08 µM.  
These data indicate that the three biological parameters that are expected for a CDK4/6 inhibitor, growth 
inhibition, Rb-dephosphorylation and G1 arrest, all occur in drug-treated cells at comparable 
 


 
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concentrations of PD 0332991. This correlation suggests that these biochemical readouts are related and a 
result of the same mechanism of action (i.e., specific inhibition of CDK4/6). 
 
1.5 
Lack of Efficacy Against Rb-Negative Tumors 
 
To ensure that PD 0332991 acts exclusively by inhibition of Rb phosphorylation, PD 0332991 was tested 
on Rb-negative tumor cells, the MDA-MB-468 human breast carcinoma and the H2009 human non-small 
cell lung carcinoma, both of which have deleted Rb. PD 0332991 had no antiproliferative activity on these 
cells at the highest concentration tested (3 µM), which is 1-2 orders of magnitude higher than the 
concentration necessary to inhibit Rb-positive tumor cells. Further evidence that the anti-tumor activity 
observed for Rb-positive tumors is due to inhibition of CDK4/6 was obtained by testing PD 0332991 in 
Rb-negative human tumor xenograft models, the MDA-MB-468 human breast carcinoma and the DU-145 
human prostate tumor. Neither tumor responded to PD 0332991.  These findings indicate that PD 
0332991 is not expected to have an anti-tumor effect in tumors that do not express Rb, and that only 
patients with tumor types that express Rb should be included in a clinical trial of PD 0332991.  
 
1.6 
 Preclinical Toxicity Data 
 
PD 0332991 has been examined in various genetic toxicology and safety pharmacology (cardiovascular, 
neurofunctional, and pulmonary) studies. General toxicology (acute, oral-escalating dose, and 2-week 
dose range-finding) studies were conducted in rats and dogs.  Pivotal toxicology studies were conducted 
in both species with PD 0332991 administered once by gavage daily for 3-weeks, followed by a 1-month 
reversal period. 
 
The primary PD 0332991 toxicities in preclinical studies are to the bone marrow, lymphoid tissues, and 
testes.  These toxicities occurred in both rats and dogs, and are consistent with cell cycle inhibition 
produced by the intended pharmacology of the drug.  Pancytic bone marrow depletion resulted in 
decreases in various hematology parameters; however, the changes were reversible following cessation of 
dosing. Reversible myelosuppression is anticipated to occur in the clinic and to be dose-limiting.  
PD 0332991 demonstrated a potential for clastogenicity in the in vitro and in vivo micronucleus assays. 
Acute intravenous administration of PD 0332991 to dogs resulted in significant pulmonary effects, 
including apnea, which were reversible.  Effects were transient, appeared related to peak plasma 
concentrations (≥2040 ng/mL), and consistent with centrally-mediated respiratory depression.  No 
changes in pulmonary function occurred at plasma drug concentrations  ≤414 ng/mL. Pulmonary changes 
were observed in rats administered PD 0332991 orally, which included rales, dyspnea, and atrophy of 
tracheal epithelium.  The clinical relevance of these effects is unknown.  Results of the Purkinje fiber and 
HERG in vitro assays, and cardiovascular study in dogs have indicated a potential for prolongation of the 
QT interval. 
 
1.7 
Phase I clinical trial 
 
There are currently 4 ongoing trials.  One study (A5481001) has been initiated to evaluate the safety of 
PD 0332991 in patients with advanced cancer.  Enrollment for this study (Protocol A5481001) has been 
completed with 74 enrolled patients and 5 active patients.  This study is evaluating two different dosing 
schedules of PD 0332991.  The first dosing regimen to be evaluated was a 3/1 schedule (3 weeks on 
treatment/1 week off treatment) and started with an initial cohort receiving 25 mg of PD 0332991 
administered orally once daily.  The dose was escalated using a new cohort for each dose level to a dose 


 
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of 150 mg once daily.  At this dose, 2/6 patients experienced DLTs related to myelosuppression.  The 
dose was reduced to 125 mg once daily for the next cohort.  This dose has been well tolerated and was 
considered the RP2D.  As per protocol, a second dosing regimen, Schedule 2/1 (2 weeks on 
treatment/1 week off treatment) was also evaluated, beginning when there was 1 DLT in the Schedule 3/1.  
The initial dose level for Schedule 2/1 was 100 mg administered orally once daily which was escalated 
using a new cohort for each dose level to 225 mg once daily.  At this dose level, 2/3 patients experienced 
myelosuppression-related DLTs and the dose was reduced to 150 mg once daily for the next cohort.  This 
dose has been well tolerated and was considered the RP2D for this schedule.  The most common adverse 
events other than those related to myelosuppression included fatigue, nausea, constipation, diarrhea, 
emesis and dyspnea; all of which had CTC Grade of 2 or less.  For Schedule 3/1, 11/41 patients exhibited 
stable disease for ≥ 16 weeks including 7 patients exhibiting stable disease for ≥48 weeks in a variety of 
tumor types.   
 
A second ongoing study (A5481002) is evaluating PET imaging modalities, target inhibition, clinical 
activity and safety of PD 0332991 in patients with previously treated mantle cell lymphoma.  PD 0332991 
is being administered on the Schedule 3/1 at an oral dose of 125 mg once daily.  At this time, enrollment 
has been completed and 17 patients have entered the study but no efficacy data are available.  So far, the 
safety profile has been similar to that observed in the dose escalation study. 
 
A third study (A5481004) is a Phase 1/2 evaluation of PD 0332991 in combination with bortezomib and 
dexamethasone in patients with refractory multiple myeloma.  Currently the Phase 1 portion of the study 
in ongoing and evaluating Schedule 3/1 with 6 patients dosed.  Five of these patients have discontinued 
from the study due to progressive disease.  The first cohort evaluated 100 gm PD 0332991, 1 mg/m2 
bortezomib and 20 mg dexamethasone.  There were 2 DLT’s due to Grade 3/4 thrombocytopenia in this 
cohort.  Thus, this cohort was closed and the second cohort (75 mg PD 0332991, 1 mg/m2 bortezomib and 
20 mg dexamethasone) was opened..  Four patients have been dosed in this cohort with one patient having 
a DLT (Grade 3 thrombocytopenia).  Enrollment into this cohort is currently ongoing. 
 
A fourth study (A5481003) is a Phase 1/2 evaluation of PD 0332991 in combination with letrozole in the 
first-line treatment of ER+ and HER2- advanced breast cancer in postmenopausal women.  The Phase 1 
portion of the study is ongoing with 3 patients currently enrolled. 
 
1.8 
Pharmacokinetics 
 
Pharmacokinetic data from the first in patient study (A5481001) indicated that PD 0332991 median time 
to peak concentration was approximately 4 hours after dosing with the exposure increasing in a generally 
dose proportional manner. The mean PD 0332991 volume of distribution was 3103 L, which is 
significantly greater than total body water (42 L), indicating that PD 0332991 extensively penetrates into 
peripheral tissues. PD 0332991 was eliminated slowly; the mean terminal half-life was 26.5 hours and the 
mean apparent oral clearance was 86.1 L/hr. PD 0332991 accumulated following repeated dosing with a 
median accumulation ratio of 2.4, which is consistent with a half-life of approximately 27 hours. Renal 
excretion was a minor route of elimination for PD 0332991 (1.7% of unchanged drug in urine). There was 
no clinically relevant effect of food on the PK of PD 0332991, indicating that PD 0332991 can be 
administered without regard to meals. 
 
1.9 
Pharmacodynamics 
 
The phosphorylation status of Rb is the most direct way to measure CDK4/6 inhibition by PD 0332991.  
By blocking Rb phosphorylation via CDK4/6 inhibition, PD 0332991 activates Rb to suppress tumor 


 
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growth by repressing expression of genes required for proliferation. Changes in Rb phosphorylation will 
be monitored in clinical samples by immunohistochemistry (ICH) using antibodies specific for certain 
phospho-epitopes of Rb, such as phospho-Ser780 Rb, that are sensitive to CDK4/6 inhibition.  To 
investigate biomarkers associated with efficacy in preclinical models, clinical samples will be assayed for 
Ki-67, a widely used marker of cell proliferation, and for the expression of certain proliferation-associated 
genes.   
 
Genetic alterations in the Cyclin D1/p16/Rb/Cdk4/6 pathway that lead to increased Rb phosphorylation 
are among the most common in human cancer. Cyclin D1 complexes with and activates CDK4/6, 
facilitating movement through the cell cycle, and its expression is frequently unregulated due to gene 
amplification, chromosomal translocation, or oncogenic signaling. p16 is a tumor suppressor protein that 
normally inhibits CDK4/6 activity; its expression is frequently repressed in tumors, often by epigenetic 
mechanisms. The net effect is to increase CDK4/6 activity leading to Rb inactivation. Expression levels of 
Cyclin D1 and p16 will be assessed in clinical samples in this study using immunohistochemistry.  
 
Genetic polymorphisms in K-ras and cyclin D1 are relatively common oncogenic alterations in human 
cancer.  Preclinical data suggests a role for K-ras genetic polymorphisms in altering sensitivity to PD 
0332991.  The relation of Cyclin D1 polymorphism to PD 0332991 is under investigation, but is known to 
affect risk and outcome of several cancers.  Genetic polymorphism in K- ras, cyclin D1, and Rb will be 
monitored in clinical tumor samples. 
 
Amplification of CCND1 and PD0332991 
Work at Pfizer has shown that breast cancer cell lines with amplified CCND1, the gene encoding cyclin 
D1, are especially sensitive to the drug.  Further, broadening the focus of investigation, they have shown 
that sensitivity in these models may also depend on the presence of mutated or inactivated p16, a negative 
regulator of the cell cycle.  And independent of these specific findings, gene expression profiling has 
identified a greater level of sensitivity for the luminal A breast cancer profile, suggesting that additional 
determinants of susceptibility can be identified.  Shapiro in a review of this field distinguished key 
consequences of cell cycle inhibition: growth arrest and cessation of proliferation that could resume upon 
removal of the inhibitor, versus induction of cell death pathways and tumor shrinkage in certain models 
(34). While either effect could be construed as beneficial in cancer treatment, the latter is likely to be 
more demonstrably effective in advanced cancer, and is a reasonable focus for future trials.  The data 
generated in this work will provide a basis for the interpretation of findings from the normal and tumor 
tissues of patients treated in these trials.    
 
Data from studies at Pfizer have implicated CCND1 amplification as a major association with sensitivity 
to PD0332991.  Further, a recent study of Beroukhim and colleagues (35) analyzed 3,131 cancer 
specimens, belonging largely to 26 histological types, and identified 158 regions of focal copy number 
alteration that were altered at significant frequency across several cancer types.  The gene that was the 
second-most frequently amplified was CCND1, and this particularly in esophageal and head and neck 
malignancies, liposarcomas and breast tumors.  Together these findings prompt the addition of a study in 
a cohort particularly likely to respond on biological grounds. 
 
1.10 
Study Rationale 
 
We have designed a Phase II trial enriched for potentially responding patients on either molecular or 
clinical grounds.  We include patients with breast cancer, esophageal, gastric cancer and colorectal cancer, 
in whom the pathway can be activated by several mechanisms, and resistant germ cell tumors.  The 


 
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inclusion of a resistant germ cell cancer population is based on our observations of significant activity in 
this population in the Phase I trial (33).  Responses or significant proportion of patients with stable 
disease in these groups will prompt a more definitive confirmatory Phase II trial.  In addition, these 
populations are likely to have tumors that take up 18F-fluorothymidine at baseline, which can then serve 
as a marker of cell cycle inhibition, and permit associations with clinical outcome, with PK, and allow the 
determination of pharmacodynamic variability.  Positive PD findings could potentially guide the future 
development of this promising agent.  In addition, given strong preclinical evidence to suggest the 
importance of the Rb pathway in HER2-overexpressing cancer cells as well as its potential role in 
secondary resistance mechanisms to HER2-directed therapy, subjects with HER2-overexpressing tumors 
may benefit from receiving  trastuzumab in combination with PD 0332991.  Thus additional patients with 
HER2-overexpressing  tumors will be allowed to receive trastuzumab concurrently with PD0332991 in 
order to obtain preliminary safety data and efficacy of the combination. 
 
 
2.  STUDY OBJECTIVES 
 
 The primary objectives of this study are: 
• To determine the response rates following treatment with PD 0332991 in the following malignancies: 
1 Metastatic breast cancer  
 
2 Metastatic colorectal cancer that harbors the Kras or BRAF mutation  
 
 
3.Advanced or metastatic esophageal and/or gastric cancer 
 
4 Cisplatin-refractory, unresectable germ cell tumors 
 
5.  Any tumor type if tissue tests positive for CCND1 amplification, CDK4/6 mutation , CCND2 
amplification OR any other functional alteration at the G1/S checkpoint. 
 
• To evaluate the safety and tolerability of PD 0332991, administered to subjects with refractory solid 
tumors 
The secondary objectives of this study are: 
• To assess the pharmacodynamic effects of PD 0332991 on tumor and non-tumor tissue  
• To investigate the relationship between selected biomarkers, PK and/or efficacy and safety 
outcomes. 
 
• To estimate the population pharmacokinetics for PD 0332991 and to correlate PK with efficacy 
outcomes. 
 
• To evaluate the preliminary safety of PD 0332991 in combination with trastuzumab in patients with 
HER-2 overexpressing tumors within the 5 cohorts under study. 
 
• To perform a Phase II evaluation of PD0332991 in a population defined as potential responders on 
the basis of CCND1 gene amplification. 
 
 


 
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3.0 
  STUDY DESIGN 
 
3.1  
Overview of Study Design 
This is a Phase II, open-label study evaluating the response rates, time to disease progression, safety, 
pharmacokinetics, and pharmacodynamics of PD 0332991 in subjects with selected solid tumors.   Up to 
30 patients in most cohorts will be accrued to the trial to determine the response rate. The cohorts are 
defined by disease type or CCND1 amplification status.  The ER-positive breast cohort will enroll up to 45 patients.  
In addition, 10 patients with HER2-positive cancer will receive PD 0332991 in combination with trastuzumab. An 
early stopping rule for lack of activity will be activated if there are no responses and no 6-month stable 
disease patients among the first 15 patients.  The estimated length of the study is 1200 months for subject 
accrual and treatment. 
A cycle of treatment is defined as 28 days.  Subjects will take PD 0332991 daily for 21 days, with cycles 
repeating every 28 days.   
 
4.0  ELIGIBILTY CRITERIA 
 
 
4.1  INCLUSION CRITERIA: 
1.  Disease Characteristics: 
All Subjects:  All subjects treated under this protocol will have histologically documented cancer of 
one of the following types:  
 
A.  Metastatic breast cancer (7 triple negative, 23 ER+ after the first 15 patients are enrolled on the non-
CCND1cohort; in addition 10 HER2+ for combination trastuzumab and PD0332991 therapy) up to 55 total            
enrollment slots 
 
B. Metastatic colorectal cancer that harbors the Kras or BRAF mutation (15-30 enrollment slots) 
 
C.   Advanced or metastatic esophageal and/or gastric cancer (15-30 enrollment slots) 
 
D.  Cisplatin-refractory, unresectable germ cell tumors (15-30 enrollment slots) 
 
E.   Any tumor type if tissue tests positive for CCND1 amplification, CDK4/6 mutation, CCND2 
amplification OR any other functional alteration at the G1/S checkpoint. (15-30 enrollment slots) 
     
2.  Biopsy Requirements:   
For Subjects with accessible disease amenable to biopsy:  A biopsy will be obtained pre-treatment and in during 
cycle 1 (while patient is receiving drug) for molecular markers of the cell cycle, and its inhibition. 
 
For all subjects: 
3.  Subjects will be > 18 years old 
4.  The subject has disease that is assessable by tumor marker, physical, or radiologic means. 
5.  The subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. 


 
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6.  The subject has adequate organ function, defined as follows 
A. Bilirubin ≤ 1.5 x the upper limit of normal (ULN) 
B. Serum creatinine ≤ 1.5 x UNL or calculated creatinine clearance ≥ 60 mL/min, and  
C. For subjects without liver metastases: alanine aminotransferase (ALT) and aspartate 
aminotransferase (AST) ≤ 2.5 x ULN  
D. For subjects with liver metastases: alanine aminotransferase (ALT) and aspartate 
aminotransferase ≤ 5 x ULN  
7. All tumors must test positive for Rb expression except:       
            A. ER positive metastatic breast tumors (data now shows all to be Rb positive.) 
            B. Any tumor type if tissue tests positive for CCND1 amplification, CDK4/6 mutation, CCND2 
amplification OR any other functional alteration at the G1/S checkpoint.  
8.  The subject has adequate marrow function, defined as follows: 
A. Absolute neutrophil count (ANC) >1500/mm3 
B. Platelets >100,000/mm3, and 
C. Hemoglobin > 9 g/dL 
9.  The subject is capable of understanding and complying with the protocol requirements and has signed 
the informed consent document. 
10. Sexually active subjects (male and female) must use accepted methods of contraception during the 
course of the study and for 3 months after the last dose of protocol drug(s). 
11. Female subjects of childbearing potential must have a negative pregnancy test at screening. Females 
of childbearing potential are defined as sexually mature women without prior hysterectomy or who 
have had any evidence of menses in the past 12 months.   
12. However, women who have been amenorrheic for 12 or more months are still considered to be of 
childbearing potential if the amenorrhea is possibly due to prior chemotherapy, antiestrogens, or 
ovarian suppression. 
 
4.2 EXCLUSION CRITERIA 
 
1. The subject has received cytotoxic chemotherapy (including investigational cytotoxic 
chemotherapy) within 3 weeks (or nitrosoureas or mitomycin C within 6 weeks) before the 
first dose of PD 0332991. .  Patients with HER2-overexpressing tumors may receive trastuzumab 
up to the date of starting therapy, and may continue to receive trastuzumab while receiving 
PD0332991. 
2. The subject has received any other type of investigational agent within 28 days before the 
first dose of study treatment.  
3. The subject has not recovered from clinically-meaningful toxicity due to prior therapy 
(i.e., back to baseline or Grade ≤ 1), with the exception of neurotoxicity and alopecia. 
4. The subject has untreated or uncontrolled brain metastases or evidence of leptomeningeal 
involvement of disease unless the subject has a teratoma in which case s/he may be eligible 
if all other eligibility criteria are met   
5. The subject has uncontrolled intercurrent illness including, but not limited to:  
a. ongoing or active infection 
b.diabetes mellitus 
c. hypertension 
d.symptomatic congestive heart failure, unstable angina pectoris, stroke or 
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       6. The subject has a baseline corrected QT interval (QTc) > 470 ms.  
 
       7.  The subject is pregnant or breastfeeding. 
 
       8.  The subject is known to be positive for the human immunodeficiency virus (HIV). Note:  
baseline HIV screening is not required 
 
       9.  The subject is unable or unwilling to abide by the study protocol or cooperate fully with  
            the investigator or designee. 
 
4.3  Concomitant Medication and Treatment 
Concomitant medications will be recorded on the CRF. 
Subjects with HER-2 positive tumors will be allowed to receive concominant trastuzumab at the 
discretion of the treating physician. Trastuzumab will be given per standard of care protocol.  If the 
subject is not currently receiving trastuzumab, a loading dose of 8mg/kg will be given intravenously on 
the first day of treatment and continued at 6mg/kg intravenously once every 3 weeks during the study 
period. 
4.4  Tumor Biopsies 
An archival tumor specimen (or a biopsy specifically to obtain tissue to establish eligibility) is required, 
and will be forwarded for testing as specified in section 6.1.  A number of patients with accessible tumor will 
have biopsies, and have the tumor biopsy divided, with one aliquot flash frozen, and the other processed for FFPE.  
Samples will be analyzed for markers of cell cycle inhibition (such as Ki67, and others) and emerging markers of 
sensitivity to cell cycle inhibition.    All patients with esophageal and/or gastric cancer will have tissue tested for 
phosphor-protein analysis, a putative marker of activated ASK1, JNK1 and 2, and c-Jun.   
4.5  Anticancer Treatment, Including Radiotherapy 
If a subject requires additional anticancer treatment, he or she must be withdrawn from the study (with the 
exception of trastuzumab for HER-2 positive tumors, palliative radiotherapy or other local therapy, which 
may be allowed during the study with the approval of the sponsor (Dr. O’Dwyer and the medical 
monitor). If anticancer treatment follows progression due to clinical deterioration determined by the 
investigator, the basis for this determination should be documented. 
4.6  Other Medications 
Subjects who develop nausea, vomiting, or diarrhea should be treated as clinically indicated. For subjects 
who have required treatment on study for nausea, vomiting, or diarrhea, prophylactic anti-emetic and anti-
diarrheal medication may be used as per standard clinical practice before subsequent doses of study drugs, 
at the discretion of the investigator.  


 
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Pain medications administered as dictated by standard practice are acceptable while the subject is enrolled 
in the study. Colony-stimulating factors should not be administered prophylactically or therapeutically: 
myelosuppression should be managed by dose reduction as specified below.. 
No concurrent investigational agents will be permitted.  No concurrent anticancer treatment will be 
permitted. 
High dose (>60mg/day) or chronic (> 3months) steroid use is not allowed during the course of this study 
as it may interfere with metabolism of the study drug.  Subjects who require anticoagulation (eg, warfarin, 
heparin compounds) while on study treatment must be monitored very closely; including frequent PT/INR 
and PTT testing. 
 
5.0  STUDY TREATMENTS 
 
5.1. Allocation to Treatment 
This is a single-center study and a centralized registration system will be used. Patients will be enrolled 
into the Phase 2 study sequentially according to their disease type. 
 
5.2. Trial Period 
Patients will receive doses of PD0332991 on an outpatient basis. Clinic visits, hematology and other 
safety laboratory tests will be performed according to the Schedule of Activities. Additional assessments 
may be performed as necessary to evaluate specific adverse events until they resolve to baseline or CTC 
Grade 1. Additional clinic visits may be required at the discretion of the investigator. 
 
5.3. Patient Dosing: 
A cycle of therapy is 28 days duration. The starting dose of PD0332991 will be 125 mg. PD0332991 will 
be administered by mouth once daily for 21 days, followed by 7 days off drug.  Repeated cycles of 
PD0332991 will be administered until patients develop progressive disease or unacceptable toxicity, 
whichever occurs first. Study treatment may also be discontinued for protocol non-compliance or 
administrative reasons detailed elsewhere in this protocol. 
 
5.3.1. Patient Dose Modifications  
PD0332991 should not be administered to patients with evidence of significant renal or hepatic 
impairment.  
For Grade 3 and 4 toxicities, treatment with PD0332991 should be withheld until the toxicity resolves to 
Grade 1 or less, then reinstituted, if medically appropriate, per Sections 5.3.1.1 and Section 5.3.2.2. 
PD0332991 doses will not be made up at a later date.  There has been extensive clinical trial experience 
with PD0332991 and has been approved for several indications, but clinical cirumstances vary 
considerably and cannot be prediected.  So as to serve the patients’ needs best  (balancing toxicity and 
benefit) there may be occasions in which the treating physician takes an approach different to that 
outlined, as in clinical practice. 
 
5.3.1.1. Dose Modification of PD 0332991 for Hematologic Toxicity 
Decisions regarding dose reduction for myelosuppression will be made based on blood counts performed 
weekly during the initial two cycles of treatment, and every 28 days at the beginning of a cycle thereafter. 
For myelosuppression requiring drug dosing to be held as specified below, the drug will be held until 
counts recover to acceptable levels (see below).  Doses of PD0332991 that are missed will not be made 


 
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up, and upon resumption of dosing, the cycle will stay on schedule.  Counts will be performed twice 
weekly during a period of dose omission, and treatment may resume when the neutrophil count (or 
platelet count) has returned to an acceptable level as defined below.  Doses will be modified according to 
the following table: 
 
 
 
Within-Cycle Dose Modifications for Hematologic Toxicity 
 
Neutrophils 
(per cubic mm) 
 
 
Platelets 
(per cubic mm) 
Action 
> 1000 
 
AND 
> 50,000 
Continue full dose 
500-999 
 
OR 
25,000 to  
49,900 
Decrease PD0332991 dose 
to 100 mg after counts 
recover to Grade 2 or better 
<500 
 
OR 
< 25,000 
Decrease PD0332991 dose 
to 75 mg after counts 
recover to Grade 2 or better 
 
 
This strategy is implemented with the recognition that exposure variability to this oral agent may be 
substantial, and that the area under the concentration-time curve is an excellent predictor of 
myelosuppression  Hence what might look like a very low dose for one patient, may in fact reflect 
significant pharmacodynamic effect. 
 
A new cycle of therapy will not commence until counts have recovered as outlined below:   
 
Dose Modifications at the Start of a New Cycle Following Hematologic Toxicity 
 
Neutrophils 
(per cubic mm) nadir 
previous cycle 
 
 
Platelets 
(per cubic mm) 
nadir previous 
cycle 
 
Action 
> 1000 
 
AND 
> 50,000 
Continue full dose 
500-999 
 
OR 
25,000 to  
49,900 
Decrease PD0332991 dose 
to 100 mg daily 
<500 
 
OR 
< 25,000 
Decrease PD0332991 dose 
to 75 mg daily 
 
Patients reduced to 100 mg whose nadirs are grade 2 or better in the subsequent cycle may be escalated to 
125 mg, and will be monitored with weekly counts for at least two cycles to ensure its tolerability.   
 
5.3.2.2. Dose Modifications of PD 0332991 for Non-Hematologic Toxicity 
In general, for Grade 3 or 4 toxicities, attributable to PD0332991, treatment should be withheld until the 
toxicity resolves to < Grade 2, then reinstituted, if medically appropriate, at a 50mg dose reduction. If 
treatment is withheld for longer than 3 weeks, discontinuation of study treatment should be considered. 


 
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Dose Limiting Non-hematologic Toxicity 
 
Action 
Any grade 3 or 4 non-hematologic toxicity 
 
Hold till resolution to Grade 2 or less, then 
restart PD0332991 at a 50 mg dose reduction 
Any Grade 2 toxicity, which in the opinion 
of the investigator, and after consultation 
with the Sponsor, is considered dose limiting 
Hold till resolution to Grade 1 or better, then 
restart PD0332991 at a 25 mg dose reduction 
Recurrent Grade 3 or 4 non-hematologic toxicity 
 
Hold till resolution to Grade 2 or less, then 
restart PD0332991 at a 50 mg dose reduction 
Inability for a patient to recover to < Grade 2 or baseline 
within 1 cycle, or miss more than 1 cycle of treatment 
Consider discontinuation of 
study treatment 
 
5.3.2.3 Dose Modification of trastuzumab and PD 0332991 for Cardiac Toxicity in patients 
receiving concurrent PD 0332991 and trastuzumab. 
   
Per standard of care, patients receiving trastuzumab and PD 0332991 will be monitored every 3 
months with an echocardiogram or MUGA.   
a. If the ejection fraction drops by >10 points, but remains normal, continue both therapies and 
repeat cardiac assessment in 4 weeks. 
b. If the ejection fraction drops by 10-20 points but the ejection fraction is >40%, continue both 
therapies and repeat cardiac assessment in 2 to 4 weeks.  If at the time of reassessment the 
ejection fraction has not improved, stop both drugs. 
c. If the ejection fraction drops by >20 points, is <40%, hold both drugs and repeat cardiac 
assessment in 2 weeks.  If the ejection fraction has improved to >45%, then restart both 
drugs.  If at the time of reassessment the ejection fraction has not improved, stop both drugs. 
 
If a subject receiving trastuzumab and PD 0332991 has symptoms suggestive of heart failure, 
cardiac assessment should be obtained immediately. 
a. If the ejection fraction drops by <10 points, continue both drugs and repeat cardiac 
assessment in 2-4 weeks. 
b. If the ejection fraction drops by >10 points but is >50%, continue both drugs and repeat 
cardiac assessment in 2-4 weeks. 
c. If the ejection fraction drops by >30 points stop both drugs. 
 
5.4. Drug Supplies 
 
5.4.1. PD0332991 
 
5.4.1.1. Formulation and Packaging 
 
PD 03322991 is formulated in 2-tone grey capsules containing 125mg, 100mg, or 75mg  of study 
medication.  The capsules can be distinguished by their size. 
 
5.4.1.2. Preparation and Dispensing 
 
Medication will be provided in non-patient specific bottles containing either 125mg, 100mg, or 75mg 
capsules.  The patient number and the protocol number should be recorded on the bottle label in the 
spaces provided.  Patients should be given a sufficient supply to last until their next study visit.   


 
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PD 0332991 is an agent that must be handled and administered with care. Patients should be instructed to 
keep their medication in the bottles provided and not transfer it to any other container.  Due to possible 
unknown hazards associated with topical and environmental exposure to experimental agents, capsules 
must not be opened and/or emptied into any vehicle for oral ingestion; capsules must be swallowed intact. 
 
5.4.1.3. Administration 
 
Patients should be instructed to swallow PD 0332991 capsules whole and not to chew them prior to 
swallowing.  No capsule should be ingested if it is broken, cracked, or otherwise not intact.  Patients 
should be encouraged to take their dose at approximately the same time each day.   
 
PD 0332991 dosing should be under fasting conditions, i.e. water only from 1 hour prior to until 2 hours 
following dosing. 
 
5.4.1.4  Compliance 
 
Study center personnel must ensure that outpatients clearly understand the directions for self-medication. 
Patients must be given a study medication diary to be completed each day that PD 0332991 is scheduled 
to be taken and be instructed how to complete it.  Unused drug and/or empty bottles should be returned to 
the site at the next study visit. 
 
5.4.1.5. Drug Storage and Drug Accountability 
 
Patients and individuals dispensing study medication must not remove capsules from the sponsor’s 
containers for dispensing or storage in any other containers.  
 
The investigator, or an approved representative (eg, pharmacist), will ensure that all study drug is stored 
in a secured area, at controlled room temperature and in accordance with applicable regulatory 
requirements. Patients are to store study medication at room temperature. 
 
5.5. Concomitant Medication(s) 
 
Treatment intended as supportive care, such as analgesics, corticosteroids, megestrol acetate, 
erythropoietin, pamidronate, vitamins, and antidepressants may be used at the investigator’s discretion. 
Prostate cancer patients may remain on luteinizing-hormone releasing hormone (LHRH) agonists while 
on study.  Antiemetics may be used at the investigator’s discretion for prevention and/or treatment of 
nausea and vomiting. Every effort should be made to ensure that nausea and vomiting are controlled, as 
these conditions may preclude a patient from taking or absorbing the oral doses of PD 0332991.  Patients 
requiring palliative radiation therapy for bone pain from a pre-existing lesion after completing Cycle 1 
may remain on study. Patients requiring radiation therapy in Cycle 1 are considered to have progressive 
disease and are to discontinue study treatment. Study medication must be interrupted while the patient 
receives a course of radiation therapy, as any potential interaction between PD 0332991 and radiation 
therapy is unknown. Such patients should be restarted on study medication after a post-radiation therapy 
recovery period of at least 1 week. Patients interrupting treatment to receive radiation therapy for ≥3 
weeks, ie, ≥3 weeks elapsed from the planned start date of the next treatment cycle, should be considered 
for discontinuation of the study, unless there is evidence of clinical benefit.  
 


 
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There have been no clinical studies to evaluate the possibility for drug interactions between PD 0332991 
and other medications.  Because inhibition of CYP3A isoenzymes may increase PD 0332991 exposure 
leading to a potential increase in toxicities, the use of known strong potent CYP3A inhibitors (such as 
ketoconazole, miconazole, itraconazole, posaconazole, clarithromycin, erythromycin, tilithromycin, 
nefazodone, diltiazem, verapamil, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, 
amprenavir, fosamprenavir, St. John’s wort and grapefruit juice) are not recommended.   
In addition, drugs that are known strong CYP3A inducers (such as phenobarbital, rifampin, phenytoin, 
carbamazepine, rifabutin, rifapentin, clevidipine, and St. John’s Wort) are not recommended. 
Concominant trastuzumab will be optional in subjects with HER2 positive tumors.  There are no expected 
overlapping toxicities with trastuzumab and PD 0332991, however, safety assessments will be carried out 
closely on these subjects.  Because of cardiac toxicity associated with trastuzumab, subjects receiving 
trastuzumab will be monitored with an echocardiogram or MUGA scan every 3 months per standard of 
care. 
 
6. STUDY PROCEDURES 
 
6.1. Screening 
All patients being considered for the study and eligible for screening must sign an informed consent for 
the study prior to any study specific procedures. The required screening assessments and laboratory tests 
are summarized in the Schedule of Activities Tables.  A historical or fresh biopsy sample will be obtained 
to evaluate the tumor for Rb expression.  This screening test for Rb will be performed in the Department 
of Pathology. Note that Rb screening is not required for eligibility for subjects with ER positive breast cancer. 
Following completion of the pretreatment assessments and confirmation of eligibility, patients may be 
scheduled to begin study treatment.   
 
6.2. Study Period 
Patients will receive doses of PD0332991 on an outpatient basis. Clinic visits, hematology and other 
safety laboratory tests will be performed according to the Schedule of Activities. Additional assessments 
may be performed as necessary to evaluate specific adverse events until they resolve to baseline or CTC 
Grade 1. Additional clinic visits may be required at the discretion of the investigator.   
 
6.3. Follow-Up Visit 
The primary reason for a patient’s discontinuation of the study medication will be clearly documented on 
the Case Report Form (CRF).  
 
Adverse events that are serious, suspected to be related to study drug, or considered significant by the 
investigator or  medical monitor must be followed after the time of therapy discontinuation until the event 
or its sequelae resolve or stabilize at a level acceptable to the investigator and the medical monitor or 
his/her designated representative. Each serious adverse event (SAE) must be reported to Pfizer in 
accordance with the terms detailed in the Penn-Pfizer Alliance 
 
Required tests and procedures for the follow up period are detailed in the Schedule of Activities. If a 
patient does not return for a scheduled visit, every effort should be made to contact the patient. In any 
circumstance, every effort should be made to document patient outcome, if possible. The investigator 
should inquire about the reason for withdrawal, request the patient to return for a final visit and follow-up 
with the patient regarding any unresolved adverse events. If the patient withdraws from the trial and also 


 
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withdraws consent for disclosure of future information, no further evaluations should be performed and 
no additional data should be collected. The Sponsor may retain and continue to use any data collected 
before such withdrawal of consent. 
 
6.4. Subject Withdrawal 
Patients may withdraw from treatment at any time at their own request, or they may be withdrawn at any 
time at the discretion of the investigator or sponsor. Reasons for withdrawal include, but are not limited to 
the following: 
 Progressive disease; 
 Unacceptable toxicity; 
 Global deterioration of health-related symptoms; 
 Protocol non-compliance; 
 Pregnancy; 
 Patient request; 
 Lost to follow-up; 
 Study termination by Sponsor. 
 
Subjects may withdraw from the study at any time at their own request, or they may be withdrawn at any 
time at the discretion of the investigator or sponsor for safety, behavioral, or administrative reasons. If a 
subject does not return for a scheduled visit, every effort should be made to contact the subject. In any 
circumstance, every effort should be made to document subject outcome, if possible. The investigator 
should inquire about the reason for withdrawal, request the subject to return all unused investigational 
product(s), request the subject to return for a final visit, 
 
If the subject withdraws from the study, and also withdraws consent for disclosure of future information, 
no further evaluations should be performed, and no additional data should be collected. The sponsor, Dr. 
O’Dwyer, may retain and continue to use any data collected before such withdrawal of consent. 
 
7. ASSESSMENTS 
 
The baseline period is from 14 days prior to the patient’s dose of PD0332991. The first treatment is Day 1 
of Cycle 1. For laboratory tests, normal ranges will be provided from the site.  Assessments required to 
evaluate the safety of PD0332991 are outlined in the following sections. 
 


 
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SCHEDULE OF EVENTS 
 
Routine screening evaluations are to be performed within 2 weeks of starting treatment.  Imaging may 
occur up to 4 weeks prior to treatment and testing for Rb expression may occur any time pre-treatment. 
 
1       Samples (1) On Cycle 1 Day 14 at pre-dose; (2) On Cycle 1 Day 15 at pre-dose, at 4 hours post-dose and 
between 7-10 hours post-dose, (2) matched with FLT-PET assessments (breast and colorectal patients only) 
except at baseline, (3) matched with tumor assessments except at baseline  
2 FLT-PET will be performed in about 15 patients with colon cancer and breast cancer only.  FDG PET 
will be performed in patients with colorectal cancer only.  The follow-up scan will be performed in 
either week 6 or 7 for each study. 
3  ECHO/MUGA will be obtained only for subjects with HER2-overexpressing tumors who will be 
receiving concominant trastuzumab.  It will be repeated every 3 months to monitor cardiac function 
per standard of care. 
 
Test 
Screening Baseline 
Week 1 
Week 2 
Week 3 
Day 1 
Subsequent 
Cycles 
Week 6 or 
7 
Informed 
Consent 
X 
 
 
 
 
 
 
PhysExam 
X 
X mini 
 
 
 
X 
 
Weight 
 
X 
 
 
 
X 
 
Height 
X 
 
 
 
 
 
 
EKG 
X 
X 
 
 
 
X (cycle 2 
only) 
 
Vital Signs/PS 
X 
X 
 
 
 
X 
 
Rb testing 
X 
 
 
 
 
 
 
CBC with diff 
X 
X 
X 
X 
X 
X  
 
OCOMP 
X 
X 
 
X 
 
X 
 
Urinalysis 
 
X 
 
 
 
 
 
PK 
 
 
 
X1 
 
 
 
PD 
 
X 
 
 
 
 
 
Optional 
Biopsy 
 
X 
 
 
X 
 
 
AE assessment 
 
X 
X 
X 
X 
X 
 
Con Meds 
X 
X 
X 
X 
X 
X 
 
Disease 
Assessment 
(scan, MRI) 
X 
 
 
 
X every 
other 
cycle 
 
 
Tumor Marker 
 
X 
 
 
 
X every 
other cycle 
 
FLT PET2 
 
X 
 
 
 
X 
 
 
FDG-PET2  
 
 
X 
 
 
 
 
 
 
 
X 
 
ECHO/MUGA3  
X 
 
 
 
 
 


 
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7.1. Physical Examination 
A complete physical examination will include the assessment of all body systems, the measurement of 
body weight, height, ECG, pulse, and assessment of ECOG performance status.  All examinations must 
be performed by qualified health care professionals. Findings of all physical examinations should be 
recorded in the source documents, and any change from baseline considered by the investigator to be 
clinically significant should be recorded as an adverse event in the case report form. 
 
7.2. Hematology and Coagulation Function 
The following hematology tests will be performed at the intervals described in the Schedule of Activities: 
hemoglobin (Hgb), hematocrit (Hct), red blood cell count (RBC), white blood cell count with 5-part 
differential (WBC w/diff), and platelet count will be collected.  Assessments will be performed within 72 
hours prior to the designated timepoints on Day 1. Flexibility (1-3 days) will be allowed around the post-
treatment blood tests for logistical reasons (eg, weekends and holidays); however, every effort should be 
made to obtain test specimens according to schedule. 
 
7.3. Serum Chemistry 
The following serum chemistry tests will be performed according to the Schedule of Activities: sodium 
(Na), potassium (K), chloride (Cl), bicarbonate (HCO3), glucose (Glu), creatinine (Cr), blood urea 
nitrogen (BUN), total bilirubin (TBili), aspartate aminotransferase (AST), alanine aminotransferase 
(ALT), lactate dehydrogenase (LDH), calcium (Ca), phosphorus (P), and alkaline phosphatase (AP). 
Assessments will be performed within 72 hours prior to the designated timepoints on day 1. Flexibility 
(1-3 days) will be allowed around the post-treatment blood tests for logistical reasons (eg, weekends and 
holidays); however, every effort should be made to obtain test specimens according to schedule. 
 
7.4. Complete Urinalysis: 
Complete microscopic urinalyses will be performed according to the Schedule of Activities on Day 1 of 
Cycle 1: color, appearance, specific gravity, pH, protein, glucose, occult blood, ketones, leukocyte 
esterase, nitrite, bilirubin, urobilinogen, and microscopic analysis of centrifuged sediment for casts, cells 
and crystals will be performed at baseline and as clinically indicated.  
 
7.5. Cardiac Monitoring 
All subjects with HER2-overexpressing tumors who will be receiving concominant trastuzumab will have 
a baseline assessment of ejection fraction with an echocardiogram or MUGA scan.  This will be followed 
every 3 months per standard of care to assess for any changes in the ejection fraction while on 
trastuzumab therapy.  Baseline ejection fraction must be at least 50% to receive trastuzumab.  PD 
0332991 and trastuzumab will be held according to section 5.3.2.3. 
 
7.6. Tumor Assessments 
All patients in this Phase 2 study will be considered evaluable for response. Baseline scans should be 
obtained within 28 days prior to enrollment. For the purpose of this study, patient’s tumor assessments 
should be re-evaluated at the end of every second cycle (approximately every eight weeks). All patients 
with responding tumors (complete or partial response) must have the response confirmed no sooner than 4 
weeks after the first documentation of response. Objective tumor response will be measured using the 
Response Evaluation Criteria in Solid Tumors (RECIST, Appendix 1).   For details, see the Schedule of 
Activities, which summarizes information on the timing of study assessments. The determination of 
antitumor efficacy will be based on objective tumor assessments made according to the system of 
unidimensional evaluation. The same method and technique should be used to characterize each identified 
and reported lesion at baseline, during the study treatment period, and during follow-up. Imaging-based 


 
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evaluation rather than clinical examination is the required technique when either could be used to assess 
the antitumor effect of the treatment. Computed Tomography (CT) scan or Magnetic Resonance Imaging 
(MRI) is the preferred method for the tumor assessment. Whenever possible, clinical evaluation of 
superficial lesions should not be used as the sole form of measurement. X-ray, ultrasound and 
radionuclide scan should not be used to measure tumor lesions. 
 
Tumor assessments can occur every 12 weeks for patients who have been receiving study drug for 18 
months or more, at the discretion of the treating physician.   
 
7.7  Pharmacokinetics of PD0332991 
 
For all patients, blood specimens for analysis of PD0332991 will be collected: (1) On Cycle 1 Day 14 at 
pre-dose; (2) On Cycle 1 Day 15 at pre-dose, at 4 hours post-dose and between 7-10 hours post-dose, (2) 
matched with FLT-PET assessments (breast and colorectal patients only) except at baseline, (3) matched 
with tumor assessments except at baseline. The actual dosing time must be collected on Cycle 1 Days 13, 
14 and 15 and on the day of the FLT-PET/tumor assessments.  All actual PK collection times should be 
documented in the CRF. 
 
Blood specimens (3 mL) to provide a minimum of 1.5 mL plasma will be collected into appropriately 
labeled tubes at times specified above. All efforts will be made to obtain the pharmacokinetic samples at 
the exact nominal time relative to dosing on Days 14 and 15, and if the patient has inadvertently taken that 
day’s dose, no sample will be drawn, and the reason noted.   
 
Following collection of 3 mL of blood into a K2EDTA tube, the tube should be inverted gently 15 times 
to completely mix whole blood with the anti-coagulant. The sample tubes should be kept on ice while 
awaiting processing and should be kept between 2 °C and 8 °C during all processing steps. Within 30 min 
of collecting the blood samples should be centrifuged at 3500 rpm (1000 x g) for 10 minutes at 4oC. A 
clean (never used) pipette should be used to transfer the plasma layer ( ~1.0 mL) into a pre-labeled 
storage cryovial and the cryovial placed upright in a storage box and stored frozen at -20 °C. 
.Samples will be analyzed using a validated analytical method in compliance with Pfizer’s standard 
operating procedures.  
 
7.8 
Pharmacodynamics 
 
Whole body PET/CT scans will be carried out on patients with colorectal cancer, and breast cancer if 
feasible, for an hour immediately following 10-15 mCi [18F]-fluoro-L-thymidine IV.  These scans will 
enable estimation of the uptake of FLT into tumor.  Surrogate values of relative uptake (SUV) will also be 
obtained.  Before PET imaging, patients will be instructed to fast for at least 6 h, and to drink 1 L of water 
before imaging to stimulate 18F-FLT excretion from the renal calyces and subsequent voiding.  
 
For injection of the radiopharmaceuticals, a venous cannula will be inserted in each patient’s forearm. All 
patients will be scanned in 8-10 positions from the skull base to the mid-thigh, with 5 min per bed 
position for emission scanning.  PET images will be iteratively reconstructed (ordered-subset expectation 
maximization).The major marker to be assessed as an indicator of cell cycle inhibition will be FLT-PET.  
The surface expression of thymidine salvage transporters is a cell cycle dependent activity that should be 
markedly reduced in the presence of an effective cell cycle inhibitor.   
 
18F-Fluoro-labelled thymidine (FLT), an analogue of thymidine and marker of proliferation, has been 
proposed as a novel PET radiotracer. In this study 18F-FLT will be used, under an Investigational New 


 
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Drug Application (IND #102,482), to assess cellular proliferation in a subset of patients with breast or 
colorectal cancer. Approximately 15 subjects will receive this investigational agent in this study. 
  
Participants receiving 18F-FLT will be brought to the Division of Nuclear Medicine and Clinical 
Molecular Imaging for administration of the agent and subsequent PET/CT imaging of the whole body.  A 
single dose of approximately 10 mCi to 15 mCi of 18F-FLT will be administered as a bolus intravenous 
injection. The injection will begin after adequate intravenous access is ensured (preferably in an 
antecubital vein).  Subjects will be encouraged to void urine immediately prior to receiving the 
investigational agent and asked to remove metal objects that may be in the field of view. At completion of 
injection, the syringe and i.v. line will be flushed with saline solution. Subjects will be monitored for 
adverse events and a whole body imaging with PET/CT will begin approximately 60 minutes following 
administration of 18F-FLT.  
  
18F-FLT PET/CT imaging 
18F PET/CT imaging from the skull base to the mid-thigh will be performed approximately 60 minutes 
after 18F-FLT administration, per standard acquisition protocols in the Division of Nuclear Medicine and 
Clinical Molecular Imaging. Attenuation-corrected PET/CT images of the whole body will be processed 
and reviewed per Division guidelines by the Nuclear Medicine Authorized User associated with this 
research study.  
 
 
7.9 Pathology Correlates 
 
All patients entered in the study will be Rb positive.  However we expect that certain tumor characteristics 
will predict for response among the diseases to be analyzed.    For breast and colon cancers, the analyses 
of cdk 4/6 and of cyclin D will be retrospective, and will attempt to identify patients who either respond 
or have prolonged stable disease.  Samples from patients with biopsiable disease will be evaluated to 
determine the degree of cell cycle inhibition achieved with PD0332991 treatment, its relationship to drug 
exposure, and to imaging markers, and will be analyzed for additional –omic studies as an approach to 
understanding markers of susceptibility to this therapy, in an exploratory fashion, and as funding plans 
evolve.   
In addition, we will look at a cohort defined by the single determinant of CCND1 amplification.  Patients 
will qualify through quantitation of gene amplification by FISH, performed by Dr Paul Zhang in the 
Department of Pathology, University of Pennsylvania.   The requirement for integral marker assay 
performance in a CLIA-approved environment makes this collaboration especially important.  The 
criterion for positivity will be a 4:1 ratio of CCND1:CEP11 control in >15% cells.  A large component of 
the study will involve screening, so to make this study practical from a budgetary as well as a patient 
perspective, we will enrich populations by selecting those for screening based on known populations with 
high incidence.  The populations to be screened will include: 
  
a.  Esophageal cancer, especially squamous cell – cohorts to be analyzed separately. 
b.  Head and neck squamous cell cancer. 
c.  Breast, especially those with luminal B expression profile, ER positivity, and high Ki67 expression. 
d.  Liposarcoma 
e.  Any histology if already known to carry the amplification 
 
In addition to CCND1, patients may enroll on this study if they express or amplify CDK4 or 6.  As there 
are currently no CLIA-approved labs to performCDK 4/6 amplification, results from the University of 
Michigan’s non-CLIA approved lab may be used until a CLIA-approved lab becomes available.  


 
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Documentation of how the test is performed, who performed the test, and the results will be obtained from 
the University of Michigan’s lab and filed in the patient’s medical record chart.  As soon as a CLIA-
approved lab becomes available at Penn or elsewhere, analysis for CDK 4/6 expression in a CLIA 
environment will be required.  Patient’s whose tissue has been tested for CDK4 mutational status at a 
CLIA certified lab may also be acceptable if adequate expression is reported.   
 
7.10 Long-term Follow-up (Germ cell tumor participants only) 
 
For participants enrolled to the germ cell tumor cohort, we will further clarify response and post-study 
treatment/progression through long term follow up via the participant’s medical record and interaction 
with treating oncologist.  Specifically, we will collect information regarding survival, date of last visit 
with oncology clinician, post-study treatments for cancer, outcome of post-study treatments for cancer, 
any cause hospitalizations, all with applicable associated dates.   
 
The medical record will be reviewed at minimum every 6 months and data associated with the above 
recorded for the purposes of the study.   
 
8 ADVERSE EVENT REPORTING 
 
8.1 Adverse Events 
The investigator must grade AEs according to the National Cancer Institute Common Terminology 
Criteria for Adverse Events Version 5.0 (CTCAE v5.0) and provide an opinion regarding the causal 
relationship of the AE to study drug.  
 
Patients will be carefully monitored for adverse events from the initial dose of study medication through 
the Follow-Up visit (Appendix C).  
 
All observed or volunteered adverse events, regardless of treatment group or suspected causal relationship 
to study drug, will be recorded on the adverse event page(s) of the case report form. 
Events involving adverse drug reactions, illnesses with onset during the study, or exacerbations of pre-
existing illnesses should be recorded.  Exacerbation of pre-existing illness, including the disease under 
study, is defined as a manifestation (sign or symptom) of the illness that indicates a significant increase in 
the severity of the illness as compared to the severity noted at the start of the trial. 
 
8.2  Serious Adverse Events 
Reporting of Serious Adverse Events.  Within 24 hours of first awareness of the event 
(immediately if the event is fatal or life-threatening), Principal Investigator will report to 
FDA and Pfizer by facsimile any Serious Adverse Event (“SAE,” as defined below) that 
occurs during the SAE reporting period (as defined below) in a Study subject assigned to 
receive the Pfizer Product or Compound (see Section 7, Pfizer Product or Compound).  
Principal Investigator will report such SAEs using a FDA MEDWATCH form and the 
Reportable Adverse Event Fax Cover Sheet provided by Pfizer.  SAEs should be reported as 
soon as they are determined to meet the definition, even if complete information is not yet 
available.   
 
a. 
SAE Definition.  An SAE is any adverse event, without regard to causality, that is life-
threatening or that results in any of the following outcomes: death; in-patient 
hospitalization or prolongation of existing hospitalization; persistent or significant 
disability or incapacity; or a congenital anomaly or birth defect.  Any other medical 


 
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event that, in the medical judgment of the Principal Investigator, may jeopardize the 
subject or may require medical or surgical intervention to prevent one of the outcomes 
listed above is also considered an SAE.  A planned medical or surgical procedure is 
not, in itself, an SAE. Also specifically excluded from this definition of SAE is any 
event judged by the Principal Investigator to represent progression of the malignancy 
under study, unless it results in death within the SAE Reporting Period.  
b. 
SAE Reporting Period.  The SAEs that are subject to this reporting provision are those 
that occur from after the first dose of the Pfizer Product or Compound through 28 days 
after discontinuation of the Pfizer Product or Compound.   
c. 
Follow-Up Information.  Institution will assist Pfizer in investigating any SAE and 
will provide any follow-up information reasonably requested by Pfizer. 
d. 
Regulatory Reporting.  Reporting an SAE to Pfizer does not relieve Institution of 
responsibility for reporting it to FDA, as required. 
 
e. 
Pfizer-Provided Training.  Pfizer will make available training material that provides 
information about the SAE reporting requirements for IIR studies.  Principal 
Investigator will review this material and share it with any Study staff engaged in the 
reporting of SAEs. 
 
Reporting Process to the IRB 
  
Principal Investigators are required to submit reports of unanticipated problems posing risks to 
subjects or others using the “Unanticipated Problems” link in the HS-ERA system.   
For reportable deaths, the initial submission to the IRB may be made by contacting the appropriate 
IRB Coordinator. IThe AE/Unanticipated Problem Form via HS-ERA is required as a follow up to the 
initial submission. 
Reporting to DSMC 
All SAEs for Penn subjects regardless of grade, attribution or expectedness must be submitted to the 
DSMC within 30 days. Unexpected deaths should be reported within 24 hours. Reports should be sent 
to the DSMC until the study is formally terminated with the IRB through Penn-CTMS. 
*IND SAFETY UPDATES/ALERTS*  
All IND Safety Updates/Alerts (sent by sponsors) with a grade 3 or higher, regardless of attribution or 
expectedness must be submitted to the DSMC within 30 days. Reports should be sent to the DSMC for 
6 months from the date the last Penn subject was treated.  
Please contact Cancer Center’s Office for Compliance with any questions on reporting.   
Medical Monitoring 
 


 
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It is the responsibility of the Principal Investigator to oversee the safety of the study at his site.  This 
safety monitoring will include careful assessment and appropriate reporting of adverse events, as noted 
above, as well as the construction and implementation of a site data and safety monitoring plan.  
Medical monitoring by an independent clinician Dr.Adam Cohen, Department of Medicine, Division of 
Hematology-Oncology will include a regular assessment of the number and type of serious adverse 
events on a periodic basis.  Dr. Adam Cohen will be provided with copies of all SAEs and and AE 
tables every 3-6 months, depending on rate of accrual.   
 
 
 
 
8.3 Abnormal Laboratory Test Results 
 
Laboratory results will be assessed for adverse events as described in CTCAE v5.0 using normal ranges 
specified for the institution’s laboratory.  
 
The results of all laboratory tests required by the protocol will be recorded in the subject’s case report 
form.  All clinically important abnormal laboratory tests occurring during the study will be repeated at 
appropriate intervals until they return either to baseline or to a level deemed acceptable by the investigator 
or until a diagnosis that explains them is made.  The criteria for determining whether an abnormal 
laboratory test result should be reported as an adverse event are as follows: 
 
1. Test result is associated with accompanying symptoms, and/or 
2. Test result leads to a change in study dosing or discontinuation from the study, significant additional 
concomitant drug treatment or other therapy, and/or 
3. Test result leads to any of the outcomes included in the definition of a serious adverse event, and/or 
4. Test result is considered to be an adverse event by the investigator or sponsor. 
 
Merely repeating an abnormal test, in the absence of any of the above conditions, does not meet 
Condition 2 above for reporting as an adverse event. 
 
8.4  Abnormal Physical Examination Findings 
 
Clinically significant changes, in the judgment of the investigator, in physical examination findings 
(abnormalities) will be recorded as adverse events. 
 
8.5  Discontinuations 
 
The reason for a subject discontinuing from the study will be recorded in the case report form.  A 
discontinuation occurs when an enrolled subject ceases participation in the study, regardless of the 
circumstances, prior to completion of the protocol. The investigator must determine the primary reason 
for discontinuation. Withdrawal due to adverse event should be distinguished from withdrawal due to 
insufficient response according to the definition of adverse event noted earlier. A discontinuation must be 
reported immediately to the Pfizer clinical monitor or his/her designated representative if it is due to a 
serious adverse event. The final evaluation required by the protocol will be performed at the time of study 
discontinuation.  The investigator will record the reason for study discontinuation, provide or arrange for 
appropriate follow-up (if required) for such subjects, and document the course of the subject’s condition. 
 
8.6   Data Safety and regulatory issues 


 
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8.6.1 Data Monitoring Plan 
 
Data will be monitored in accordance with the Cancer Center’s Clinical Trials Scientific Review and 
Monitoring Committee (CTSRMC) Plan, approved by NCI during the Core Grant’s most recent review.  
This plan requires that each investigator submit a study-specific plan for how s/he is reviewing data (see 
attached).  In addition, this plan calls for an internal audit by the Cancer Center’s Data Safety Committee 
(a subcommittee of the CTSRMC) of all data at least once per year for each study.  Finally, toxicities, in 
particular are reviewed and discussed at each weekly meeting of the Developmental Therapeutics 
Program. 
 
8.6.2  Data Plan 
 
Case Report Forms (CRFs) developed by the Developmental Therapeutics Program will be modified to 
collect data points specific to meeting the data needs of a study.  The CRFs will be reviewed and 
approved by the PI.  These CRFs will then be converted to electronic CRFs that will be available only to 
select members of the Developmental Therapeutics Program.  The CRFs include an audit trail to ensure 
data integrity.  Source documents will be developed to ensure that each data point to be collected can be 
captured.  In addition, all data enter via EPIC will be printed out and maintained in a shadow chart to 
ensure its ongoing access to the research staff and its auditors.  A designated Research Coordinator with 
experience in data management will be responsible for ensuring that the data are generated, collected and 
transcribed appropriately. 
 
On a quarterly basis, as specified by the Penn/Pfizer Alliance, study progress, but no specific patient data 
will be provided to Pfizer.   
   
9  DATA ANALYSIS/STATISTICAL METHODS 
All data for this trial will be summarized using tabulations and frequencies for dichotomous variables and 
means, medians, variances, and ranges for continuous variables.  Summaries will be stratified by disease 
site, and characteristics of each population will be compared descriptively. 
 
The response rate of interest in this trial will be 15% for each of the indications tested.   If we observe at 
least one response in the first fifteen subjects, we will enroll a further 15 subjects, for a total of 30.  
Observing zero responses in the first 15 subjects would exclude response rates as low as 15% with a 90% 
one-sided upper confidence bound.  If at least three responses are observed in a total of 30 evaluable 
patients, we will conclude that the drug is active and merits further study; if the true response rate is 15% 
or greater, three or more responses will be observed with a probability of at least 85%.  If the true 
response rate is only 3%, three or more responses will be observed with a probability of only 6%.  In 
addition to responses of CRs and PRs, disease stabilization will be considered: if the proportion of 
patients with disease stabilization for 6 months exceeds 20%, we will conclude that the drug may warrant 
further investigation in that indication.  With n=30 subjects and a true response rate of 15%, the expected 
confidence interval width is ± 10.7% around the estimated proportion.  Moreover we will have 90% 
power to detect any unforeseen adverse effect that occurs in at least 7% of cases. 
 
A subject who completes at least 2 full cycles of therapy and undergoes the required imaging at 8 weeks is defined 
as evaluable for response.  A subject who is not evaluable for response may be replaced. 
 


 
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We will compute confidence intervals for selected safety and exploratory variables. Adverse event terms will be 
standardized using the Medical Dictionary for Regulatory Activities (MedDRA) and tabulated by system organ 
class and preferred term. Laboratory parameters will be summarized using mean change in value and/or shift in 
category since baseline. 
 
Subjects evaluable for response is defined as subjects who complete at least 2 full cycles of therapy and undergo the 
protocol required imaging at 8 weeks. By extension subject not evaluable for response may be replaced such that 
the accrual goal is reflective of evaluable subjects within the respective cohort. 
 
Population Pharmacokinetic Analysis and PK/PD Modeling 
Pharmacokinetic data from this study may be analyzed using mixed-effect modeling approaches.  The 
intent of this analysis will be to build upon a basic population pharmacokinetic model for PD 0332991 
and to determine the inter-individual and residual variability in population PK parameters of interest. If 
data permit, PK/PD correlations to investigate any causal relationship between PD 0332991 exposure and 
efficacy outcomes will be explored. 


 
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