Phase I/II Randomized Study of Combination Immunotherapy With or Without Polysaccharide Krestin (PSK®) Concurrently With a HER2 ICD Peptide-Based Vaccine in Patients With Stage IV Breast Cancer Receiving HER2-Targeted Monoclonal Antibody Therapy
Phase: PHASE1
Conditions: HER2/Neu Positive, Recurrent Breast Carcinoma, Stage IV Breast Cancer
Sponsor: University of Washington

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UNIVERSITY OF WASHINGTON 
DEPARTMENT OF MEDICINE 
DIVISION OF ONCOLOGY 
 
 
Protocol 135 
 
Phase I/II Randomized Study of Combination Immunotherapy With or Without Polysaccharide 
Krestin (PSK®) Concurrently with a HER2 ICD Peptide-Based Vaccine in Patients with Stage IV 
Breast Cancer Receiving HER2-Targeted Monoclonal Antibody Therapy 
 
 
Investigators 
 
 
 
William R. Gwin, MD 
Assistant Professor 
Division of Oncology, UW 
(206) 221-5956 
Mary L. Disis, M.D. 
Professor of Medicine 
Division of Oncology, UW 
(206) 616-1823 
 
 
 
 
John Liao, M.D. 
Assistant Professor 
GYN/ONC, UW 
(206) 543-3915 
 
 
 
 
Andrew Coveler, M.D. 
Assistant Professor 
Division of Oncology, UW 
(206) 221-7509 
Sasha Stanton, M.D. 
Acting Instructor 
Division of Oncology, UW 
(206) 685-8849 
Doreen Higgins, B.S.N. 
Research Nurse 
Division of Oncology, UW 
(206) 616-9538 
Jennifer Childs, M.P.H. 
Research Coordinator 
Division of Oncology, UW 
(206) 616-2305 
 
 
 
 
Ted Gooley, Ph.D 
Biostatistician 
Fred Hutchinson Cancer 
Research Center 
(206) 667-6358 
 
Emergency (24 hour) pager:  
William R. Gwin, M.D.  
(206) 314-1279 
 Mary L. (Nora) Disis, M.D.   (206) 427-8700 
IRO received 08/23/21
FHCRC IRB Approval  
SEP 03 2021 
Document Released Date

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Table of Contents 
  Page 
Synopsis 
3 
List of Appendices 
6 
List of Abbreviations 
7 
Introduction 
8 
Background 
8 
Study Rationale 
13 
Objectives 
13 
Experimental Agent Preparation 
HER2 ICD Peptide-Based Vaccine 
14 
GM-CSF 
14 
Vaccine Dose Compounding and Dispensing 
15 
PSK Product and Placebo 
15 
Experimental Agent Accountability 
16 
Inclusion and Exclusion criteria 
16 
Experimental Design 
Study Design 
17 
Sample Size 
18 
Outcome Measures 
18 
Plan of Treatment – Arms 1 and 2 
Pre Screen  
19 
Initial Evaluation 
19 
Monthly Immunizations 
20 
Evaluation after Final Vaccine 
20 
Management of Potential Study Risks 
20 
Patient Withdrawal from Study  
21 
Evaluation and Management of Toxicity 
21 
Dose Modifications 
22 
Accrual and Criteria for Premature Study Termination 
22 
Statistical Consideration 
22 
Administrative Considerations 
25 
References 
26 
Appendices 
29 

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SYNOPSIS 
Title  
Phase I/II Randomized Study of Combination Immunotherapy With or Without 
Polysaccharide Krestin (PSK®) Concurrently with a HER2 ICD Peptide-Based 
Vaccine in Patients with Stage IV Breast Cancer Receiving HER2-Targeted 
Monoclonal Antibody Therapy 
Study Population 
Patients with Stage IV HER2+ breast cancer who have been treated with 
definitive therapy and are receiving maintenance HER2-targeted monoclonal 
antibody therapy (defined as trastuzumab monotherapy or combination 
trastuzumab and pertuzumab therapy); and are currently without evidence of 
disease (NED) or have stable-bone only disease.   
Rationale 
Hot-water extracts of mushrooms have been used in traditional medicine in China, 
Japan, and other Asian countries for centuries.  PSK was isolated in Japan from 
the Trametes versicolor mushroom in the 1960s and has been studied in more 
than 48 clinical trials for various types of cancers.  Randomized clinical trials have 
shown low toxicity profile and improved survival in breast cancer patients treated 
with PSK.   Pre-clinically, in vitro studies have shown PSK to be a potent TLR-2 
agonist that activates dendritic cell maturation, activates human NK cells, induces 
pro-inflammatory cytokine/chemokine secretion and potentiates trastuzumab-
mediated ADCC against breast cancer cells.  PSK has also been shown to 
potentiate antitumor effects of anti-HER2 mAb therapy in neu-transgenic mice.  
We hypothesize that the use of PSK will augment the innate anti-tumor immune 
responses specifically NK-cell activity.  Additionally, PSK could potentially 
augment HER vaccine induced Th1 immunity and collectively the combination 
therapy could result in enhanced eradication of micrometastatic disease which 
could prevent relapse in patients with optimally treated HER2+ Stage IV Breast 
Cancer. 
Oral PSK at 3000 mg/day has been shown to be safe with and without 
chemotherapy in breast cancer.  Moreover, combination immunotherapy with 
HER2-directed immunotherapy (HER2 ICD vaccination and HER2-targeted 
monoclonal antibody therapy) has been shown to be safe and immunogenic.  
However, the safety of PSK when added to HER2-directed immunotherapy is less 
well known and warrants further investigation.  This study will allow us to better 
define the safety and immunomodulatory effects of this combined immunotherapy 
regimen. 
Objectives 
Primary 
1. To evaluate the safety of PSK when given with HER2-directed immunotherapy. 
 
Secondary  
1. To evaluate the effect of PSK on NK cell functional activity when given with 
HER2-directed immunotherapy. 
 
Tertiary  
1. To investigate the effect of PSK when given with HER2-directed 
immunotherapy on: 
a. Serum levels of pro-inflammatory cytokine and/or chemokines. 
b. Intermolecular epitope spreading. 
c. Serum TGF-β levels. 
d. Progression free survival (PFS) and overall survival (OS). 
Study Design 
A phase I/ II double-blind randomized two-arm clinical trial designed to examine 
the safety and immune effects of PSK when given as combination HER2-directed 
immunotherapy (a HER2 ICD peptide vaccine and HER2-targeted monoclonal 
antibody therapy) in patients with Stage IV HER2+ breast cancer.  Eligible 
patients will be enrolled and randomized in equal numbers to one of 2 arms (15 
patients per arm). 

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Arm 1:  
HER2 ICD vaccine + HER2-targeted monoclonal antibody therapy* + 
Placebo: Patients will receive HER2 ICD peptide vaccine administered 
concomitantly with HER2-targeted monoclonal antibody therapy and placebo 
product. 
Arm 2:  
HER2 ICD vaccine + HER2-targeted monoclonal antibody therapy* + PSK: 
Patients will receive HER2 ICD peptide vaccine administered concomitantly with 
HER2-targeted monoclonal antibody therapy and PSK.  
*Trastuzumab (or trastuzumab and pertuzumab) will be administered per 
standard of care by patient’s primary oncologist.  
Number of Patients   A total of 30 patients will be enrolled and randomized in equal numbers to one of 
2 arms with PSK or placebo. 
Outcome Measures 
 
Primary Endpoint: 
1. Evaluation of safety of PSK when administered with HER2-directed 
immunotherapy.  Evaluation of safety and systemic toxicity will include 
physical examination and clinical labs performed at various timepoints.  
Toxicity grading will be evaluated per CTEP CTCAE 4.0 and monitoring of 
adverse events will be done per FDA and NCI guidelines.  Additionally, 
stopping rules for accrual to study will become operational if there is sufficient 
evidence of excessive toxicity. 
Secondary Endpoint: 
1.  Evaluation of the effect of PSK on NK cell functional activity.  PBMC will be 
obtained and cryopreserved at baseline (prior to start of PSK) and then at 4, 
8, and 16 weeks.  PBMC will subsequently be thawed and used to determine 
the effect of PSK on NK cell functional activity specifically, induction of IFN-
gamma production and CD107a expression in NK cells, via flow cytometry.  
Augmentation of NK cell activity is defined by a 2-fold increase in NK cell IFN-
gamma production and CD107a expression from baseline after 4 weeks of 
oral administration of PSK.  
Tertiary Endpoints:  
1. Assessment of the effect of PSK on pro-inflammatory serum cytokine and/or 
chemokines when administered with a HER2-directed immunotherapy.  Serial 
blood draws will be done before initial dose of PSK (time 0) and 30 min, 4 
hours, 
and 
24 
hours 
post-initial 
PSK 
dose 
to 
assess 
serum 
cytokines/chemokines (i.e., IL-6, IFN-γ, and TNF-α) via Luminex analysis.  
Serum cytokine/chemokine levels at different timepoints post-PSK will be 
compared to baseline levels.  Serum will be cryopreserved and subsequently 
be thawed at time of analysis. 
2. Assessment of the effect of PSK on intermolecular epitope spreading when 
administered with HER2-directed immunotherapy.  IFN-γ ELISPOT assay will 
be used to evaluate T cell precursor frequency to specific breast tumor 
antigens (HER2, IGFBP-2, TOPO-IIα, p53, and PRAME) at various 
timepoints.  A positive immune response will be defined as a post-vaccination 
T cell precursor frequency >1:20,000 antigen-specific PBMCs.  In patients 
with a baseline precursor frequency >1:20,000, a positive post-vaccination 
immune response will be defined as a 2-fold increase in antigen-specific 
PBMC.  PBMC will be cryopreserved and subsequently be thawed at time of 
analysis. 
3. Assessment of the effect of PSK on serum TGF-β levels when administered 
with HER2-directed immunotherapy.  Serum levels of TGF-β will be assessed 
with ELISA at various timepoints to determine incidence and magnitude of 

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serum TGF-β reduction pre- and post-combination immunotherapy.  Serum 
will be cryopreserved and subsequently be thawed at time of analysis. 
4. Evaluation of PFS and OS.  Evaluation of PFS and OS will be followed and 
compared between the treatment arms.  Though not statistically powered to 
this endpoint, large differences if observed between the treatment groups will 
be noted and described. 

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Appendices 
 
Appendix A 
 
Zubrod Performance Status Scale  
 
Appendix B  
  
Schedule of Events 
 
Appendix C  
  
Data Safety Monitoring Plan 

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Abbreviations: 
ADCC 
antibody-dependent cell-mediated cytotoxicity 
ALND 
axillary node dissection 
APC  
antigen presenting cells 
BUN 
blood urea nitrogen 
CBC 
complete blood count 
CFC 
cytokine flow cytometry 
CMP 
Complete metabolic panel 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
CTL 
cytotoxic T lymphocytes 
DC 
dendritic cell 
DTH 
delayed type  hypersensitivity 
DSMP 
Data Safety Monitoring Plan 
ECD 
extracellular domain of HER2 
FISH 
fluorescence in situ hybridization 
rhuGM-CSF rhu granulocyte macrophage colony stimulating factor 
GMP 
good manufacturing practice 
HER2 
HER-2/neu 
HPLC 
high pressure liquid chromatography 
ICD 
intracellular domain of HER2 
id. 
intradermal 
IFNγ 
interferon-gamma 
IHC 
immunohistochemistry 
LVEF 
left ventricular ejection fraction 
MBC 
Metastatic breast cancer 
MHC 
major histocompatability complex 
MUGA 
multigated acquisition 
NED 
no evidence of disease 
NK 
Natural Killer Cell 
OS 
overall survival 
PBMC 
peripheral blood mononuclear cells 
PSK 
Polysaccharide Krestin 
PFS 
progression free survival 
Th 
T helper cells 
TLR 
Toll-like receptor agonist 
TNF 
Tumor necrosis factor 

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UPN 
unique patient number 

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1. Introduction 
Patients with advanced breast cancer have been shown to have defects in anti-tumor immunity including 
impairment of natural killer (NK) cells.[1-3]  NK cells are key components of innate immunity which play a 
fundamental role in tumor immune surveillance. Recent findings in breast cancer patients show peripheral 
blood and tumor-infiltrating NK cells to have tumor-promoting alterations in their activating and inhibitory 
receptors which correlate with decreased cytotoxic NK cell function.[3]  Additionally, inhibitory factors in 
the tumor microenvironment including TGF-beta (TGF-β) have been shown to be directly involved in the 
impairment of NK cells’ phenotype and functions.  It is postulated that invasive breast tumors escape NK 
cell antitumor immunity via a strong inhibitory microenvironment resulting in tumor progression and 
recurrence.  Thus, novel approaches to improve NK cell function and overcome tumor cell-mediated NK 
suppression are needed. 
Toll-like receptor agonists (TLRs) are a group of pathogen recognition receptors that can recognize the 
pathogen associated molecular patterns (PAMPs) from virus, bacteria, and fungus resulting in initiation of 
innate immunity.  Several TLR agonists have been shown to activate NK cells, either directly or indirectly 
via dendritic cells (DC).[4-6]  Activation of NK cells can result in direct NK cell mediated cytotoxicity of tumor.  
Activated NK cells also secrete proinflammatory cytokines and chemokines which regulate and direct 
immune responses.[7]  Specifically, activated NK cells secrete IFN-γ which can promote maturation and 
differentiation of DCs toward a Th1 profile and drive the activation of adaptive immunity.  Our group has 
been studying the immune modulatory and anti-tumor potential of Polysaccharide Krestin (PSK), a hot 
water extract from Tremetes Versicolor mushroom as a means to enhance NK cell function via stimulation 
of TLR2.  Our pre-clinical studies have shown that PSK: (1) is a selective TLR2 agonist with potent 
antitumor effects which are dependent on enhanced PSK-induced NK and CD8 T cells activity[8], (2) can 
activate human NK cell function and enhance trastuzumab-mediated antibody-dependent cell-mediated 
cytotoxicity (ADCC)[9], (3) can induce NK cell and gamma-delta T cell secretion of multiple inflammatory 
cytokines including IFN-γ resulting in DC maturation and activation[8, 9] and (5) may also work as a vaccine 
adjuvant to significantly augment antigen-specific T cell immunity.  Collectively, these findings suggest 
the potential of PSK to augment antitumor immunity via stimulation of DCs and NK cells and also to 
enhance the therapeutic effect of trastuzumab and HER2 vaccination in breast cancer patients.   
We hypothesize that the use of PSK will enhance the innate anti-tumor immune responses specifically 
NK-cell activity and result in prolonged upregulation of IFN-γ production; this could also result in 
stimulation of adaptive Th1 immunity.  Thus, we have designed a phase I/II randomized study of 
combination immunotherapy with oral PSK administered concurrently with trastuzumab and HER2-
targeted peptide vaccination.  Our primary and secondary objectives are to assess the safety of PSK as 
a component of combination immunotherapy and to evaluate effect of PSK on NK cell functional activity.  
Additionally, we will investigate if PSK stimulates circulating proinflammatory cytokine/chemokines, 
enhances HER2 vaccine-induced T cell immunity (epitope spreading) and decreases serum TGF-β. 
2. Background 
A. Natural killer (NK) cell deficiency has been found in patients with breast cancer.  NK cells are 
part of the innate immunity that plays a fundamental role in tumor immune surveillance.  As surveillance 
cells in the tumor microenvironment, DCs and NK cells have been shown to infiltrate tumors and monitor 
the presence of different antigen derived from tumors.[10]  Thus, NK cells limit not only the emergence of 
viral infections but also of cancers by surveillance of “missing-self” and “induced-self” ligands, and by 
direct recognition of pathogen-associated molecules.  Unfortunately, NK cell maturation and cytotoxic 
function is frequently impaired in breast cancer patients.[1-3]  Moreover, NK cell activity of IFN-γ production 
has been shown to be significantly impaired in invasive breast tumors, especially in advanced stage 
disease.[3] 
The activation of NK cells is regulated by a balance of signals transduced by activating and inhibitory 
receptors expressed on the surface of NK cells.  More recently, it has been shown that breast tumor cells 
can alter NK cell function through modulation of their surface receptors.3  Specifically, expression of 
activating NK cell receptors (i.e., NKp30, NKG2D) were significantly decreased while expression of 
inhibitory receptors (i.e., NGK2A) were increased in both peripheral blood NK cells (p-NK) and breast 
tumor infiltrating NK cells (Ti-NK); and this alteration was correlated with decreased NK cell function, 
especially cytotoxicity.3  Moreover, impairment of Ti-NK cytotoxicity was more pronounced when 
compared to that of peripheral NK cells.  Additionally, in situ levels of inhibitory factors such as TGF-β and 

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regulatory T cell infiltrates correlated with the NK cell impaired phenotype and functions.  These data 
support the notion that breast tumors are able to model their environment to evade NK cell antitumor 
immunity which likely contributes to tumorigenesis.  Thus, further investigation of immunomodulators such 
as PSK to restore NK cell maturation and activity within an immunosuppressive tumor microenvironment 
and prevent breast cancer progression and/or recurrence is warranted. 
B. Mushroom extract PSK is a TLR2 agonist that activates human NK cells.  Hot-water extracts of 
mushrooms have been used in traditional medicine in China, Japan, and other Asian countries for 
centuries.  Among other properties, the anticancer and immunomodulatory effects of certain mushroom 
extracts have been described and studied in depth in the lab and in clinical trials, mainly in Asia.  PSK 
was isolated in Japan from the Trametes Versicolor mushroom in the 1960s and has been studied in more 
than 48 clinical trials, mainly for colorectal, gastric, esophageal, lung, and breast cancer.  Based on the 
findings of randomized PSK clinical trials which showed a low toxicity profile and improvements in patient 
survival, PSK is commonly used as adjuvant or neoadjuvant treatment with or without chemotherapy for 
patients with colorectal, breast, and gastric cancer in Japan.[11-14]  
TLRs play a crucial role in the innate immune response and the subsequent induction of adaptive immune 
responses against microbial infection or tissue injury.[15]  Specifically, TLRs are a group of pathogen 
recognition receptors that can recognize the pathogen associated molecular patterns (PAMPs) such as 
dsRNA or ssRNA, unmethylated DNA, or lipopeptide from virus, bacteria, and fungus.  TLRs can induce 
strong anti-tumor activity by regulating the functions of the immune cells that infiltrate the tumor 
microenvironment.  The potential of TLR agonists, such as polyI:C (TLR3), resiquimod (TLR7/8), and CpG 
(TLR9), to activate NK cells, either directly or indirectly via dendritic cells (DC), have been shown in both 
pre-clinical and clinical studies.[4-6]  Our group has been studying the immune modulatory and anti-tumor 
potential of PSK.  Our research studies have demonstrated that PSK is a potent TLR2 agonist (Fig. 1).  
Pre-clinically, in vitro studies have shown that PSK can activate human NK cells to produce IFN-γ and 
lyse tumor cells; and induce secretion of pro-inflammatory cytokines and chemokines (e.g., IL-12, TNF-α, 
MIP-1α, and MIP-1β).   
NK cells represent a highly specialized lymphoid population with potent cytolytic activity against tumor or 
virus infected cells.  There are two subpopulations of NK cells, the CD56bright NK cells which are mainly 
responsible for cytokine production and the CD56dim NK cells that are mainly responsible for the cytotoxic 
effect of NK cells.[16]  Our preclinical studies have shown that PSK can activate both subpopulations of NK 
cells and stimulates both the IFN-γ production and cytotoxicity of NK cells (Fig. 2).[9]   
 
In a recent Phase 1 study of Trametes versicolor (Tv) in women with breast cancer conducted by Standish 
et al, NK cell activity increased to approximately 2-fold of baseline after 4 weeks of oral administration of 
Tv.[17]  Thus, the immune measure of PSK-induced NK cell activity in this study will be assessed with 
functional NK flow cytometry assays used in our previously published papers and at similar time points as 
described in Section 7.C.2. (Outcome Measures).[18]   
 
1
10
100
1000
0
1
2
3
PSK (µg/mL)
TLR4
TLR2
SEAP Activity (OD650 nm)
 
   A 
     
B 
 
PBS 
PSK 
K562
12.5
25
50
100
0
20
40
60
80
PBS
PSK
*
*
*
E:T ratio
% Specific Lysis
CD56bright 
 
CD56dim 
 
 
 
 
Figure 1.  PSK selectively activates TLR2.  
HEK cells transfected TLR2 or 4 were 
stimulated with serial dilutions of PSK (0.5-
1000 µg/mL, 24 hr). PSK also had no effect 
on TLR3, 5, 7, 8, 9 (data not shown).    
 
Figure 2.  PSK stimulates IFN-γ production and augments the cytotoxicity  of 
NK cells.  (A) Intracellular staining showing that PSK induces IFNγ production 
by NK cells.  (B) PSK-stimulated PBMC have enhanced lysis against K562.  
Data shown is representative of results from 3 donors. 
CD56 
IFN-γ 

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C. PSK enhances trastuzumab mediated ADCC and may improve its clinical efficacy. Trastuzumab 
is a humanized anti-HER2 monoclonal antibody (mAb), and is the first HER2-targeted therapy approved 
by FDA.  Trastuzumab has significantly advanced the clinical management of patients with HER2+ breast 
cancer by prolonging disease-free survival in patients with locally advanced breast cancer, and 
progression-free and overall survival in patients with metastatic breast cancer.[19, 20]  Trastuzumab inhibits 
tumor cell growth through multiple mechanisms including signaling blockade and downregulating the 
HER2/neu receptor.  One of the major mechanisms is believed to be ADCC, in which the tumor cells are 
coated with trastuzumab and then lysed by immune cells via binding of Fc gamma receptor (FcγR) to the 
Fc portion of the mAb.[21]  Increase in tumor infiltrating NK cells after trastuzumab therapy has been found 
in human breast cancer biopsy samples[22, 23] and FcγR gene polymorphism can impact the clinical 
response to trastuzumab.[24]  NK cells constitutively express FcγRIIIA (CD16) and are the major effectors 
of ADCC.[25]  Therefore the function of NK cells may impact the efficacy of ADCC and clinical response to 
trastuzumab.[26]   
In our pre-clinical studies, we have found 
that 
PSK 
significantly 
enhanced 
trastuzumab-mediated ADCC against two 
breast cancer cell lines, SKBR3 and MDA-
MB-231.  Furthermore, we showed that 
combination of oral PSK and a HER2-
targeted mAb therapy in mice resulted in 
enhanced anti-tumor effect.[18]  Patients 
enrolled to this study will have metastatic 
breast cancer which has been optimally 
treated to a state of NED (no evidence of 
disease) and therefore off cytotoxic 
chemotherapy.  However, our patient 
population 
will 
continue 
use 
of 
maintenance trastuzumab monotherapy 
or 
combination 
trastuzumab 
and 
pertuzumab therapy as dictated per standard of care.  Although not a study outcome, combination of PSK 
with trastuzumab could potentially enhance NK-cell-mediated ADCC of residual microscopic disease (i.e., 
HER2+ tumor cells) in our study patients.    
D. PSK can activate DC and augment antigen-specific T cells when used as a vaccine adjuvant. 
TLR2 is highly expressed on DC, and treatment with PSK can induce DC maturation and secretion of 
multiple inflammatory cytokines, including TNF-γ, IL-12p40, and IL-12p70.[8, 9]  Furthermore, PSK-induced 
IFN-γ produced by NK cells and gamma-delta T cells can reciprocally activate DC.  Therefore, we 
hypothesize that PSK may work as vaccine adjuvant to augment antigen-specific T cells.  Our pre-clinical 
studies in a mouse model of vaccination with ova peptide antigen p323 has shown that PSK as a vaccine 
adjuvant significantly enhanced ova-specific T cell immunity.  Specifically, BALB/c mice were given a 
vaccine of OVA p323-339 peptide at the dose 0.5 mcg per mouse concurrently with an intradermal (ID) 
adjuvant of either PSK (1000 mcg), GM-CSF (5 mcg), or PBS.  For two days following initial injection, 
adjuvant was given alone once per day and animals were sacrificed on Day 4.  Draining lymph nodes 
(dLN) were harvested for evaluation of DC activation and antigen-specific T cells.  dLNs from mice that 
received GM-CSF or PSK were enlarged compared to dLNs from mice in the PBS control group (Fig. 4A); 
and the percentage and number of CD11c+ DC were significantly increased in GM-CSF and PSK treated 
mice per FACS analysis.  The expression of activation markers, CD80, CD86, MHCII, and CD40 were 
also up-regulated in DC isolated from dLNs from PSK treated animals (Fig. 4B).  The number of ova 
antigen-specific T cells, as evaluated in an IFN-γ ELISPOT assay was also significantly increased in mice 
that received PSK as adjuvant (Fig. 4C).  Although PSK was given ID in these experiments, we 
hypothesize that oral PSK may also enhance the effect of vaccination.  Indeed, a recent publication by 
Tanaka et al has shown that combining peptide vaccine with oral ingestion of Lentinula endodes mycelia 
extract enhances anti-tumor activity in B16 melanoma-bearing mice.[27]   
SKBR3
12.5
25
50
100
0
20
40
60
80
100
PSK
PBS
E:T ratio
% Specific Lysis
 
MDA-MB-231
12.5
25
50
100
0
10
20
30
40
PBS
PSK
E:T ratio
% Specific Lysis
 
Figure 3. PSK enhances trastuzumab-mediated ADCC in lysing of 
SKBR3 and MDA-MB-231 breast cancer cells. PSK or PBS-stimulated 
PBMCs were incubated with 3000 target cells at the indicated E:T ratio. 
Shown are specific lysis at each E:T ratio (mean±sem of triplicate wells). 
A 
PBS 
GM-CSF 
PSK 
   B 
CD80          CD86         MHCII         CD40  
C

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E. PSK may augment vaccine-induced HER2 Th1 immunity in optimally treated metastatic breast 
cancer patients.  Disease relapse in optimally treated HER2+ breast cancer patients who are in complete 
remission suggest the presence of persistent residual microscopic disease not eradicated by systemic 
therapy.  One approach to the eradication of residual subclinical disease in this high-risk population is the 
development of tumor vaccines that target HER2, an immunogenic biologically relevant protein.  
Generation of vaccine-induced HER2-specific Type I inflammatory CD4+ T helper immunity (Th1) could 
result in immunologic eradication of residual HER2+ tumor cells and potentially prevent disease relapse 
or tumor spread.  Recent evidence indicates that Type I immunity which is associated with the production 
of high levels of IFN-γ enhances cross priming, the primary method by which immunity is generated 
against cancer, at the site of cancer.  Moreover, vaccine induced Th1 immunity has the potential to 
generate epitope spreading, a broadening of the immune response to multiple potential antigens in the 
tumor which may result in more efficient kill of residual tumor cells by T cells.  This is particularly important 
as impaired IFN signaling with associated T cell activation defects have been identified in breast cancer 
and may be a contributing factor to decreased Th1 immune responses to breast cancer vaccines.   
As previously described, our pre-clinical studies have shown that PSK can induce DC maturation and 
secretion of multiple inflammatory cytokines that result in antigen-specific T cell immunity.  Additionally, 
growing evidence indicates that NK cells have mechanisms to efficiently promote maturation and 
differentiation of DCs toward a Th1 profile.  Specifically, activated DCs in the tumor microenvironment are 
able to take up tumor antigens and migrate to lymph nodes where they present their antigens to CD8+ T 
cells and CD4+ T cells which then undergo clonal expansion and are able to infiltrate and destroy tumor 
in an antigen specific manner.  Thus, NK cell activation and resultant IFN-γ production indirectly promote 
adaptive antitumor T cell responses via activated DCs.[10]  Similarly, activated DCs induce potent NK cell 
activation.  Together, NK cell and DC activation are key in the priming process of CD8+ and Th1 immunity 
and development of adaptive immunity.[10]  Recognizing the importance of NK cells and their role in 
stimulating adaptive immunity, recent immunotherapeutic strategies such DC vaccination have focused 
on overcoming impaired Th1 immunity via NK cell activation.  Specifically, Baek et al, showed that DC 
vaccines when given with IL-12 were able to induce antigen specific immunity; and NK cell activation with 
reduction of inhibitory immunity.[28, 29]  In a follow-up study, Qi et al, demonstrated the ability of DC 
vaccination to promote secretion of Th1 cytokines and significantly increase the number of peripheral 
blood NK cells resulting in tumor specific CD8+ T cell expansion and further differentiation into IFN-γ 
producing effector cells.[29]   
We have previously demonstrated our ability to induce tumor-specific immunity and also epitope 
spreading in patients with HER2+ breast cancer.[30, 31]  Moreover, epitope spreading was associated with 
potential survival benefit in vaccinated patients.[32]  Key elements to inducing HER2-specific Th1 immunity 
and epitope spreading in the majority of HER2+ breast cancer patients were the use of vaccines which 
elicit Th1 immunity given in combination with GM-CSF as adjuvant and our targeted patient population 
(optimally treated patients to state of NED). The addition of immunomodulatory agents such as PSK as 
adjuvants to vaccines may help enhance, sustain, and skew the immunogenicity of antigens toward a Th1 
phenotype via robust NK cell activation, allowing for development of robust and durable vaccine-induced 
Th1 immunity; and potentially prevention of cancer recurrence.  Thus, we propose to investigate if PSK 
when given orally with HER2 peptide based vaccination and trastuzumab can enhance both development 
and magnitude of HER2 vaccine-induced Th1 T cell immunity specifically, epitope spreading.  
 
 
 
PSK 
 
 
PBS 
Figure 4.  PSK as vaccine adjuvant stimulates dendritic cell (DC) activation in vivo and enhances antigen-specific T cells. (A) 
Representative pictures of draining lymph nodes (dLN) from mice vaccinated with ova peptide with PBS, GM-CSF or PSK as 
adjuvant.  (B) The expression of DC activation markers, CD80, CD86, MHCII, and CD40 on DC in dLN from mice received 
ova plus PSK (empty histogram) or PBS control (shaded histogram). (C) The number of spots per well in IFN-γ ELISPOT 
assay evaluating ova-specific T cells using dLN cells from mice received ova plus PBS, GM-CSF or PSK.   
PBS
GM-CSF
PSK
0
50
100
150
200
*
**
Adjuvant Treatment
Spots per well

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F. PSK may enhance NK cell activation by inhibiting TGF-β production in the tumor 
microenvironment.  TGF-β is a hormonally active polypeptide that inhibits the proliferation of normal 
epithelial cells, and enhances matrix formation and angiogenesis.[33]  Most epithelial cancer cells lose 
responsiveness to TGF-β induced growth inhibition.[34, 35]  TGF-β is secreted by both breast cancer cells 
and the tumor stroma resulting in increased proliferative capacity, enhanced epithelial-mesenchymal 
transition (EMT); and subsequent increased metastatic potential of tumors.[36, 37]  TGF-β, a potent 
immunosuppressive cytokine acts in the tumor microenvironment to blunt immune-surveillance via 
multiple mechanisms, including suppression of NK cells.  Additionally, TGF-β is secreted by T regulatory 
cells which function to inhibit the development of adaptive immunity to “self-antigens” such as tumor 
antigens.[38]  Moreover, elevated levels of serum TGF-β have been found to be related with disease 
progression and worse prognosis in breast cancer patients.[39]  Recent studies have shown that inhibitory 
molecules in the tumor microenvironment such as TGF-β directly contribute to impairment of NK cell 
phenotype and function.  As described above, Mamessier et al showed that expression of activating NK 
cell receptors were significantly decreased while expression of inhibitory receptors were increased in both 
peripheral blood and breast tumor infiltrating NK cells; and stroma-derived inhibitory factors particularly 
TGF-β were involved in driving the impaired NK cell activity.  Additionally, increased in situ levels of TGF-
β and regulatory T cell infiltrates correlated with worse NK cell impairment.  Thus, inhibition of the TGF-β 
via immunomodulation of the tumor microenvironment may restore NK cell activity and result in 
development of robust tumor antigen specific Th1 immunity.  Previous studies have suggested that PSK 
may modulate the biological activity of TGF-β and thus restore robust NK cell activity.  Additionally, 
enhanced NK cell activity with subsequent increased IFN-γ production may result in decreased TGF-β 
production in the tumor microenvironment.  In a more recent study, PSK was shown to inhibit the Smad 
pathway, the major regulator of TGF-β signaling.[40]  Treatment with PSK inhibited the expression of 
several TGF-β pathway target genes and prevented TGF-β1-induced EMT.   
Studies by our group have demonstrated that circulating levels of TGF-β are significantly elevated in sera 
from breast cancer patients compared to controls; and that vaccine-induced HER2 specific IFN-γ T cell 
responses specifically epitope spreading, were associated with reductions in serum TGF-β levels.  
Additionally, the magnitude of the epitope spreading T cell response elicited with active vaccination was 
inversely correlated with TGF-β levels assessed in serum throughout immunization.  As described in 
Section 13 (Statistical Considerations) our previous study data suggest a modest decrease in serum TGF-
β levels among patients who received HER2 ICD vaccination and trastuzumab alone.  Therefore we aim 
to investigate if administration of PSK with HER2 vaccination and trastuzumab results in enhanced 
downregulation of TGF-β expression in the tumor and augmented NK cell induced IFN-γ production; and 
subsequent decrease in detectable circulating serum levels of the cytokine. 
G. Concomitant administration of pertuzamab and trastuzumab has been shown to be safe and 
effective in treating HER2+ metastatic breast cancer (MBC).  Pertuzumab is a recombinant humanized 
monoclonal antibody that binds to the extracellular dimerization domain II of HER2, and inhibits 
heterodimerization of HER2 with other HER family members, including EGFR, HER3, and HER4.  
Blockade of HER2-HER3 likely represents the most relevant action of.[41]  In pre-clinical models, 
pertuzumab when given in combination with trastuzumab, is associated with a more complete HER2 
blockade.  Additionally, the treatment combination is more effective in HER2-positive tumor xenografts 
than when either antibody is given alone.[41]  Moreover, enhanced efficacy of the combined use of 
trastuzumab and pertuzumab when given with docetaxel has now been demonstrated in several clinical 
studies in HER2+ MBC.[42]  
Over the past 2 years, the first-line treatment of HER2+ MBC has been changing and now also includes 
other HER2 targeted agents specifically pertuzumab (perjeta).  In 06/2012, the FDA approved the 
combination of docetaxel and pertuzumab/trastuzumab for the treatment of HER2+ MBC.  This was based 
on data from the CLEOPATRA study, a large Phase III trial enrolling 808 patients which showed the 
combination of docetaxel + pertuzumab/trastuzumab improved progression free survival (PFS) by 6 
months compared to docetaxel + placebo/trastuzumab.[43]  Given the PFS benefit, oncologists started 
using the triple regimen more readily.  As a result, HER2+ MBC patients started receiving indefinite 
concomitant pertuzamab and trastuzumab as maintenance therapy once docetaxel was stopped.  More 
recently, final results from CLEOPATRA (reported 02/2014) showed that first-line treatment with docetaxel 
+ pertuzumab/trastuzumab in patients with HER2+ MBC significantly improved overall survival (OS) when 

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compared to docetaxel + placebo/trastuzumab.  Median OS was 56.5 months (pertuzumab arm) vs 40.8 
months (placebo arm) with difference of 15.7 months.   
Extensive cardiac surveillance was conducted in CLEOPATRA and treatment with docetaxel + 
pertuzumab/trastuzumab did not increase the rate of left-ventricular systolic dysfunction (LVSD) when 
compared to docetaxel + placebo/trastuzumab.  Specifically, the incidence of LVSD (all grades) was 8.6% 
in placebo arm vs 5.4% in pertuzumab arm.  Additionally, the incidence of symptomatic LVSD was 1.8% 
in placebo arm vs 1.2% in pertuzumab arm.  Lastly, incidence of LVEF decline to <50% and by ≥10% 
points from baseline was 7.4% in placebo arm vs 4.6% in pertuzumab arm; and LVEF recovery to ≥50% 
was 89.3% in placebo arm vs 88.9% in the pertuzumab arm.  Altogether, no increased cardiotoxicity was 
observed in patients receiving concomitant pertuzumab and trastuzumab with docetaxel when compared 
to concomitant placebo and trastuzumab with docetaxel.  Given the significant OS benefit, and the 
acceptable toxicity data, it has been recommended that the new standard of care for first-line treatment 
of HER2+ MBC include docetaxel + pertuzumab/trastuzumab.   
3. Study Rationale  
The proposed oral dose of PSK (3000 mg/day) in this study has been demonstrated to be safe with and 
without chemotherapy in large randomized breast cancer studies.[44, 45]  In a review of PSK use and 
adverse events (AEs), 114 (1%) AEs were reported from a total of 11,300 individuals who used PSK alone 
or with other medical treatments (Table 1).  Principal AEs were diarrhea (0.25%), nausea (0.24%), 
vomiting (0.15%), and anorexia (0.15%).  These data are taken from the Japanese Ministry of Health and 
Welfare and are in the PSK Package Insert.  Severe PSK-related AEs have not been reported since 
clinical trials began in Japan in the 1970s thus, PSK appears to be extremely well tolerated.   
Table 1.  Adverse Event Summary for PSK Dose (3g/day) Based on 11,300 Individuals 
Symptomatic 
Diarrhea (0.25%) 
Nausea (0.24%) 
Vomiting (0.15%) 
Anorexia (0.15%) 
Hives (0.1%) 
Stomach discomfort (0.1%) 
Hematologic 
None 
Combination immunotherapy with HER2-directed immunotherapy has also been shown to be both safe 
and immunogenic in our previous studies.  However, the safety of PSK when added to HER2-directed 
immunotherapy is less well known and warrants further investigation.  This study will allow us to better 
define the safety and immunomodulatory effects of this regimen on the tumor microenvironment and 
systemic immunity.   
4. Objectives 
A. Primary objectives: 
1. To evaluate the safety of PSK when given with HER2-directed immunotherapy. 
B. Secondary objectives: 
1. To evaluate the effect of PSK on NK cell functional activity when given with HER2-directed 
immunotherapy. 
C. Tertiary objectives: 
1. To investigate the effect of PSK when given with HER2-directed immunotherapy on: 
a. Serum of pro-inflammatory cytokine and/or chemokines. 
b. Intermolecular epitope spreading. 
c. Serum TGF-β levels. 
d. Progression free survival (PFS) and overall survival (OS). 
 
 
 
5. Experimental Agent Preparation 
A. HER2 ICD peptide-based vaccine  

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The synthetic HER2 peptides to be used in the vaccine (Table 2) are 15 amino acids in length with an 
amino acid sequence identical to the normal HER2 sequence.  This HER2 vaccine has been evaluated in 
a previous study (see Background) and is covered by BB IND 6524 (Sponsor: Mary L Disis, M.D.).   
1. Peptide synthesis and characterization: 
The peptides are manufactured by Multiple Peptide Systems, San Diego, CA. using standard solid 
phase synthesis techniques employing Boc chemistry.  Amino acids are analyzed by TLC and 
polarimetry prior to use.  The peptides are cleaved from the solid support using anhydrous hydrogen 
fluoride and conventional techniques.  Each peptide is purified by HPLC using a Waters Delta-prep 
system with a gradient of acetonitrile (containing 0.1% trifluoroacetic acid) in water (also containing 
0.1% trifluoroacetic acid).  The peptides are lyophilized to dryness before being passed over an anion 
exchange column to exchange the trifluoroacetate for acetate.  GMP procedures are followed for the 
manufacture of these sequences.  After formulation is complete, the peptides are characterized by 
mass spectrometry to confirm their identity, by HPLC for purity using two different elution buffers, and 
by amino acid analysis for identity and peptide content.  The water content of each peptide was 
determined by the Karl Fischer procedure and the data obtained used to verify the mass balance of 
the peptides.  
These peptides are vialed as the HER2 ICD by the Fred Hutchinson Cancer Research Center, 
Biologics Product Facility (a GMP facility). 
2. Peptide formulation and stability: 
Prior to formulation, the solubility of each peptide in 10 mM sodium acetate buffer (pH 4.0) is 
determined.  Three peptides were made up in a 10 mM sodium acetate buffer (pH 4.0).  The stability 
of each individual peptide in the acetate buffer, as well as the peptide mixtures, is monitored by HPLC 
and mass spectrometry.  The dissolved peptides are stored at -20 ± 5˚C prior to use.  Microbial and 
sterility testing are conducted to ensure safety.  The peptide formulation is labeled to accurately reflect 
the product identity, concentration, lot number and fill date.  These labels are attached to vials 
immediately post-fill. 
B.  GM-CSF 
Recombinant human GM-CSF (rhuGM-CSF; Sargramostatin, LEUKINE) will be used as adjuvant admixed 
with HER2 vaccine, obtained by commercial suppliers and stored at their specifications.  GM-CSF is a 
growth factor that supports the survival, clonal expansion, and differentiation of hematopoietic progenitor 
cells including dendritic APCs.  In general, the use of GM-CSF is associated with little toxicity; and rhuGM-
CSF, when given IV or subcutaneously is well tolerated at doses ranging from 50-500 mcg/m2/day.  
Severe toxicity is extremely rare in patients treated with rhuGM-CSF.  For the current study, rhuGM-CSF 
will be used at a total injection dose of 100 mcg.  
Administration of rhuGM-CSF may aggravate fluid retention in patients with pre-existent edema, capillary 
leak syndrome, or pleural or pericardial effusions.  In some patients with pre-existing renal or hepatic 
dysfunction, elevation of the serum creatinine or bilirubin and hepatic enzymes has occurred during 
rhuGM-CSF administration.  Dose reduction or interruption of rhuGM-CSF administration has resulted in 
a decrease to pretreatment values.  Occasional transient and reversible supraventricular arrhythmia has 
been reported in uncontrolled studies, particularly in patients with a previous history of cardiac arrhythmia.  
Stimulation of marrow precursors with rhuGM-CSF may result in a rapid rise in white blood cell count.  If 
rhuGM-CSF is being used for the purpose of hematopoietic reconstitution, dosing should be stopped if the 
ANC exceeds 20,000/cm3.  The dose being used in this study should not affect ANC levels.  RhuGM-CSF 
may stimulate the growth of myeloid malignancies; therefore caution must be exercised in its use in these 
malignancies or myelodysplastic syndromes.  RhuGM-CSF is a sterile, white, preservative-free lyophilized 
powder in 250 mcg vials.  Reconstituted rhuGM-CSF will be admixed and administered with the HER2 
vaccine. 
C.  Vaccine Dose Compounding and Dispensing 
Table 2.  HER2 ICD peptides 
HER2 ICD Vaccine 
Sequence 
p776-790 
GVGSPYVSRLLGICL 
p927-941 
PAREIPDLLEKGERL 
p1166-1180 
TLERPKTLSPGKNGV 

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The HER2 ICD Vaccine is supplied in single-use vials as a sterile, frozen solution.  Each single-use 1.1 
ml vial contains equal volumes of ICD peptides, p776-790, p927-941, p1166-1180 (625 mcg of each 
peptide per ml) for a final concentration of 1.9 mg/ml total ICD peptide mix. The ICD vaccine will be 
supplied as frozen vials and each vial will carry a label bearing the drug identification and conditions for 
storage. The University of Washington Investigational Drug Pharmacy (UW IDS) will prepare and dispense 
the drug for administration per their standard operating procedure as follows:  
1. Reconstitute rhuGMCSF 250mcg with 0.5ml sterile preservative free water and add rhuGMCSF 
125mcg/0.25ml to a sterile empty 2ml vial. 
2. Thaw HER2 ICD Vaccine 1.9mg/ml and withdraw 1 ml and add to vial containing the rhuGMCSF.  
Total volume in vial is 1.25ml.  Rotate vial gently to mix.  
3. Total dose is HER2 ICD 1500mcg and GMCSF 100mcg in 0.99ml.  Divide dose into 3 syringes, 
containing 0.33ml each for intradermal injection. 
D. PSK Product and Placebo  
This PSK and Placebo characterization and storage are covered by BB IND 114546 (Sponsor: Leanna 
Standish, PhD, ND). The PSK® and Placebo are to be stored at room temperature (59ºF - 86ºF). 
 
1. Description and Composition of PSK® Clinical Drug Product 
The PSK® clinical drug product is a granular powder, light tan to dark brown in color, which is 
packaged in unit dose aluminum pouches (Table 3) by First Priority in Ferndale, WA.  There are no 
excipients, other botanical substances, synthetic or highly purified drugs, biotechnology-derived 
drugs, or other naturally derived drugs in the drug product.  Once reconstituted with water, a clear to 
slightly turbid brownish solution is obtained for oral administration.   
 
2. Description and Composition of Placebo: 
The placebo is a granular powder, light tan to dark brown but mottled with white in color, which is 
packaged in unit dose aluminum pouches by First Priority in Ferndale, WA.  There are no botanical 
substances, synthetic or highly purified drugs, biotechnology-derived drugs, or other naturally derived 
drugs in the drug product (Table 4).  Once reconstituted with water, a clear to slightly turbid brownish 
solution is obtained for oral administration. 
 
3. Description and Composition of Container Closure System: 
The primary container closure system for PSK® clinical drug product and placebo is a laminated 
aluminum pouch.  The aluminum pouch is composed of components that are commonly used in the 
pharmaceutical industry for solid, oral dosage form products.  The container closure system is selected 
based on comparable specifications to that used for the marketed drug product. 
4. Description and Composition of Dosage Preparation Kit:   
A shaker cup/lid is provided to facilitate preparation of the oral dosage solution.  Briefly, the PSK® 
clinical drug product or placebo is emptied into the shaker cup, which has already been filled with 
approximately 4-8 oz. of warm water.  The lid is then applied to seal the cup and shaken vigorously to 
dissolve the powder.  The liquid preparation is to be consumed within 15 minutes.  After the initial 
Table 3. Quantitative Composition of the PSK® Clinical Drug Product in Each Unit Dose Pouch 
Component 
Source 
Amount 
Function 
PSK® Bulk Drug Product 
Kureha 
1.5 g 
Active Ingredient 
Table 4.  Description and Composition of Placebo 
Component 
Source 
Amount 
Function 
Mannogem®™ 2080, 
Granular Mannitol, USP 
SPI Pharma (Wilmington, DE) 
1.2 g 
Bulking Agent 
Caramel Color 643a 
D. D. Williamson & Co., Inc. 
(Louisville, KY) 
0.3 g 
Colorant 
aClass III caramel color used in sauces, gravy, beer, baking, other foods. 

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preparation is consumed, additional warm water is added, shaken and consumed to rinse the cup and 
ensure the entire dose has been taken. 
E. Experimental Agent Accountability  
The HER2 vaccine, GM-CSF and PSK will be managed by the UW IDS.  UW IDS will be responsible for 
study drug disposition (drug receipt, dispensing, transfer or return).  Once IDS receives notification of the 
subject visit they will begin thawing the vaccine and prepare the appropriate number of syringes ready for 
injection as described in Section 5.C.  UW IDS will deliver the HER2 vaccine, PSK and/or the placebo to 
the Clinical Research Center, where patients will be seen.  The disposal of clinical trial materials that are 
in UW IDS will be incinerated under a state contract through an EPA licensed facility.  Unused PSK 
pouches will be destroyed. 
As part of our Data Safety Monitoring Plan (DSMP) (see Appendix C) an independent monitor will be 
assigned by the Fred Hutchinson Cancer Research Center Clinical Trials Support Office to monitor our 
study twice per year, which includes a review of the UW IDS dispensing logs for the vaccine and PSK.   
6. Subject Selection  
A. Inclusion criteria 
1. Patients with stage IV HER2+ breast cancer treated to: 
a. No evidence of disease (NED), or  
b. Stable bone only disease after definitive therapy. 
2. HER2 overexpression by IHC of 2+ or 3+ in the primary tumor or metastasis; or documented gene 
amplification by FISH analysis.  IHC ≤ 2+ must have HER2 gene amplification documented by FISH.  
3. Patients must continue HER2-targeted monoclonal antibody therapy dosing per standard of care 
through the entire study period (one year). 
a. HER2-targeted monoclonal antibody therapy is defined as either trastuzumab monotherapy, or 
trastuzumab and pertuzumab combination therapy administered per standard of care. 
4. Patients must be at least 21 days post cytotoxic chemotherapy prior to enrollment. 
5. Patients must be at least 28 days post immunosuppressants prior to enrollment. 
6. Patients must be at least 28 days from use of any mushroom supplements (examples: turkey tail, 
reishi, maitake, shiitake,) and agree to withhold them for the entire study period (one year). 
7. Patients on bisphosphonates and/or endocrine therapy are eligible. 
8. Patients who are having sex that could lead to pregnancy must agree to contraceptive use during the 
entire study period. 
9. Patients must have Zubrod Performance Status Score of ≤ 2 (Appendix A). 
10. Patients must have recovered from major infections and/or surgical procedures, and in the opinion of 
the investigator, not have significant active concurrent medical illnesses precluding study treatment. 
11. Laboratory values must be as follows and performed within 30 days of enrollment: 
a. WBC ≥ 3000/mm3 
b. Hgb ≥ 10 g/dl  
c. Serum creatinine ≤ 2.0 mg/dl or creatinine clearance > 60 ml/min 
d. Total bilirubin  ≤ 1.5 mg/dl 
e. SGOT ≤ 2.5 times the upper limit of normal 
12. Patients must be at least 18 years of age 
13. Patients must have adequate cardiac function as demonstrated by normal left ventricular ejection 
fraction (LVEF) ≥ the lower limit of normal for the facility on MUGA scan or echocardiogram (ECHO) 
within 3 months of enrollment. 
 
B. Exclusion criteria  
1. Patients with any of the following cardiac conditions: 
a. Restrictive cardiomyopathy 

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b. Unstable angina within 6 months prior to enrollment 
c. New York Heart Association functional class III-IV heart failure  
d. Symptomatic pericardial effusion. 
2. Patients with any contraindication to receiving rhuGM-CSF based products. 
3. Patients with any clinically significant autoimmune disease requiring active treatment. 
4. Patients receiving any concurrent immunosuppressants. 
5. Patients who are pregnant or breast-feeding. 
6. Patients who are simultaneously enrolled in other treatment studies. 
7. Patients who have received a previous HER2 breast cancer vaccine. 
8. Known hypersensitivity reaction to mushroom products. 
Subjects must meet all of the listed criteria in order to be eligible for study.  
7. Experimental Design 
A. Study design 
This will be a phase I/II double-blind randomized two-arm clinical trial designed to examine the safety and 
immune effects of PSK when given as combination immunotherapy with a HER2 ICD peptide vaccine in 
patients with Stage IV HER2+ breast cancer receiving HER2-targeted monoclonal antibody therapy.  
Eligible patients will be enrolled and randomly assigned in equal numbers to one of two treatment arms 
(15 patients per arm) shown below.  However, stagger enrollment of 2 weeks will occur for the first 2 
patients in each arm.  
Arm 1: 
 
HER2 ICD vaccine + HER2-targeted monoclonal antibody therapy* + Placebo: Patients will 
receive HER2 ICD peptide vaccine administered concomitantly with HER2-targeted monoclonal 
antibody therapy and placebo product. 
Arm 2: 
 
HER2 ICD vaccine + HER2-targeted monoclonal antibody therapy* + PSK: Patients will receive 
HER2 ICD peptide vaccine administered concomitantly with HER2-targeted monoclonal antibody 
therapy and PSK.  
*Trastuzumab (or trastuzumab and pertuzumab) administered per standard of care by patient’sprimary 
oncologist.  
All study patients will receive the HER2 ICD vaccine administered monthly for a total of 3 vaccines in 
addition to HER2-targeted monoclonal antibody therapy (administered per standard dosing guidelines).  
Additionally, patients randomized to Arm 1 will receive continuous placebo product given orally and 
patients randomized to Arm 2 will receive continuous PSK given orally at 3000 mg/day (1500 mg twice 
daily before meals).  Both the placebo product in Arm 1 and PSK in Arm 2 will be started on the day of the 
first HER2 vaccine and continued for a total of 4 months.  All patients will be followed for immune response 
at designated timepoints as shown in the patient treatment schema below. 
Patient Treatment Schema 
 

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B. Sample size 
The study will accrue a maximum of 30 subjects: 15 subjects per arm as described in Section 13 
(Statistical Considerations). 
C. Outcome measures 
1. Primary Endpoints: 
a. Evaluation of safety of PSK when administered with HER2-directed immunotherapy.  Evaluation 
of safety and systemic toxicity includes physical examination and clinical labs performed at 
baseline, before each vaccination, and 1 month after the 3rd vaccine.  Toxicity grading will be 
evaluated per CTEP CTCAE 4.0.  Type and grade of toxicities noted during treatment will be 
summarized.  Additionally, stopping rules for accrual to the study will become operational if there 
is sufficient evidence of excessive toxicity as described in Section 12.    
2. Secondary Endpoints: 
a. Evaluation of the effect of PSK on NK cell functional activity.  PBMC will be obtained and 
cryopreserved at baseline (prior to start of PSK) and then at 4, 8, and 16 weeks.  PBMC will 
subsequently be thawed and used to determine the effect of PSK on NK cell functional activity 
specifically, induction of IFN-γ production and CD107a expression in NK cells, via flow cytometry.  
Augmentation of NK cell activity is defined by a 2-fold increase in NK cell IFN-γ production and 
CD107a expression from baseline and after 4 weeks of oral administration of PSK.   
3. Tertiary Endpoints: 
a. Assessment of the effect of PSK on pro-inflammatory serum cytokine and/or chemokines when 
given with HER2-directed immunotherapy.  Serial blood draws will be done at baseline before the 
initial dose of PSK (time 0) and 30 min, 4 hours, and 24 hours after the initial dose of PSK to 
assess serum cytokines/chemokines (i.e., IL-6, IFN-γ, TNF-α) via Luminex analysis.  Serum 
cytokine/chemokine levels at different timepoints post-PSK will be compared to base-line.  Serum 
will be cryopreserved and subsequently be thawed at time of analysis. 
b. Assessment of the effect of PSK on intermolecular epitope spreading when given with HER2-
directed immunotherapy.  PBMC will be obtained and cryopreserved prior to the 1st vaccine and 
at 1 and 9 months after the 3rd vaccine (months 4 and 12) by IFN-γ ELISPOT.  PBMC will 
subsequently be thawed and used to determine T cell precursor frequency specific for tumor 
antigens, HER2, IGFBP-2, TOPO-IIα, p53, and PRAME.  A positive immune response will be 
defined as a T cell precursor frequency >1:20,000 antigen-specific PBMCs.  Development of a 
positive immune response after treatment begins will constitute augmentation in patients without 
pre-existent immune responses.  In patients with pre-existent immune response to the tumor 
antigens, augmentation is defined as a final response to tumor antigen >2 times the baseline level.  
Epitope spreading will be defined as the generation of significant immunity to the HER2 ECD as 
the patients will receive an ICD vaccine.   
c. Assessment of the effect of PSK on serum TGF-β levels when given with HER2-directed 
immunotherapy.  Serum TGF-β levels will be assessed via ELISA prior to the 1st vaccine 
(baseline), and 1, and 9 months after the 3rd vaccine (months 4 and 12) to determine the incidence 
and magnitude of reduction of serum TGF-β levels pre- and post-combination immunotherapy.  A 
decrease in serum TGF-β levels will be defined as a reduction of ≥ 25% from baseline value to 
post-vaccination values.  Serum will be cryopreserved and subsequently be thawed at time of 
analysis.  
d. Evaluation of PFS and OS.  PFS and OS will be followed and compared between study arms.  This 
study will not enroll sufficient numbers of subjects to give statistical power to this endpoint however, 
large differences if observed between the treatment groups (PSK + HER2-directed immunotherapy 
versus Placebo + HER2-directed immunotherapy) will be noted and described.  PFS and OS will 

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be defined as time from first vaccine to time of first event.  While on study, patients will be followed 
for disease progression by their primary oncologist per conventional practice standards.  
Information on patient disease status will be obtained biannually from their primary oncologist for 
up to 3 years after enrollment.    
8. Plan of Treatment - Arms 1 and 2 
The following section outlines the patient evaluation and treatment schedule prior to and during study. 
A. Pre Screen  
Potential subjects will be screened for eligibility by a study team member.  Standard-of-care procedures, 
done prior to signing consent, can be used for eligibility assessment as long as they are conducted within 
required windows (Section 6).  During this evaluation a pathology report will be obtained to ensure a 
patient’s tumor is HER2 positive as described in Section 6. 
B.  Initial evaluation (may be performed up to 2 weeks prior to initial vaccine visit) 
1. Sign consent form before initiating any study procedures and eligibility is confirmed. 
2. The clinical trial procedures will occur at the UW CRC.  Patients, who meet the inclusion/exclusion 
criteria after consent, will be assigned a unique study numbers and randomized to either PSK or 
placebo at time of study visit #1 – Day 1.  Following an algorithm developed by the study statistician, 
a randomization table will be generated and provided to an un-blinded IDS pharmacist.  Patients, 
clinicians, and study staff will be blinded to treatment assignment. 
a. Arm 1: Patients will be assigned to the placebo agent 
b. Arm 2: Patients will be assigned to PSK 
3. Medical history and complete physical examination which includes weight, vital signs, symptom 
assessment, Zubrod scoring, medication/supplement changes (Appendix A).   
4. Urine pregnancy testing will be performed on female patients of childbearing potential.  If a patient is 
found to be pregnant, she will not be able to participate in the study.  
5. A tetanus-diphtheria vaccine (Td) will be administered as a positive control if patient has not received Td 
immunization within the last six months.  Patients who have experienced any of the following described 
under a. and b. below will not receive the Td vaccine but will continue all other study related treatment and 
procedures.  
• 
Patients that have ever had a life-threatening allergic reaction after a dose of any tetanus or 
diphtheria containing vaccine, OR have had a severe allergy to any part of a tetanus or diphtheria 
vaccine. 
• 
Patients with epilepsy or another nervous system problem, severe pain or swelling after any vaccine 
containing diphtheria or tetanus, or ever had Guillain Barré Syndrome (GBS). 
6. Clinical labs (baseline):  
a. Complete blood count which includes a differential and platelet count  
b. Comprehensive metabolic panel (CMP) which includes electrolytes, creatinine, blood urea 
nitrogen (BUN), AST, ALT, alkaline phosphatase, and total bilirubin.  
7. Research blood includes:  
a. 200 mls for immunologic monitoring, including, cytokine kinetics and an assessment of single 
polymorphisms and T cell function that may contribute to PSK responsiveness.  
8. The research staff will instruct each patient on the mixing and administration of study drug using 
“dummy” pouches.  The study staff will ensure that they do not witness the preparation of the tea in 
order to preserve the study blind. 
9. 
Immediately after the 1st vaccine, patients will take a total dose of PSK 3000 mg/placebo while in the 
UW CRC. 
a. 30 minutes (+15 min) after the placebo/PSK dose, 10 mls of blood will be drawn for cytokine 
kinetics 
b. 4 hours (+/-15 min), after their first dose of placebo/PSK dose, 10 mls blood will be drawn for 
cytokine kinetics 

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10. Day 2 
a. 24 hours (+/-30 min) after finishing their first dose of placebo/PSK, patients will return to the UW 
CRC for an additional 10 mls blood draw for cytokine kinetics 
b. After this blood draw, patients will start taking the placebo/PSK at 1500 mg orally BID (total daily 
dose = 3000 mg) 
c. Patients will be given a sufficient amount of placebo/PSK which will equal a 30 day supply + 7 
additional days as needed, in order to provide continuous dosing until the subsequent study visit  
d. Patients will be given a monthly tea diary 
C. Monthly immunizations 
1. The following procedures and tests will be done prior to each vaccination: 
a. Physical examination which includes weight, vital signs, symptom/toxicity assessment and 
medication/supplement changes. 
b. Urine pregnancy test in patients capable of child bearing (patients found to be pregnant will not 
receive further study treatment). 
c. Clinical labs: CBC and CMP.   
d. Research blood: 40 mls for immunologic monitoring 
2. Monthly vaccines will be administered intradermally (i.d.) on the arm or leg for 3 months. 
a. Patients with axillary lymph node dissection (ALND) will have vaccine administered to the 
contralateral arm.  Patients with bilateral ALND will have vaccine administered in the thigh.  As 
much as possible each vaccine dose will be given within the same draining lymph node site. 
b. Vaccines may be scheduled 28 + 7 days apart. 
3. Post-vaccination monitoring: Patients will be observed on the UW CRC for minimum of 60 minutes 
and vital signs will be documented. 
4. Arm 1: Patients will be given a 1-month supply + 7 additional days of placebo agent and a monthly tea 
diary.  
5. Arm 2: Patients will be given a 1-month supply + 7 additional days of PSK (1500 mg twice daily) and 
a monthly tea diary. 
6. Collect any unused PSK pouches for destruction.  
Should patients show evidence of disease progression, they will be removed from the study if their 
treatment conflicts with the eligibility criteria (e.g., concurrent administration of chemotherapy, immune 
modulators, steroids, etc.). 
D. Evaluation after the final vaccine 
1. One month after the final vaccination (approximately month 4) patients will undergo: 
a. A physical examination which includes weight, vital signs, symptom/toxicity assessment and 
medication/supplement changes.  
b. Clinical labs: CBC and CMP 
c. Research blood draw of 200 mls for immunologic monitoring 
d. Arm 1: Patients will be given a 1-month supply of placebo agent and a monthly tea diary. 
e. Arm 2: Patients will be given a 1-month supply of PSK (1500 mg twice daily) and a monthly tea 
diary. 
2. Two months after last vaccine (approximately month 5) patients will undergo research blood draw of 
40 mls for immunologic monitoring. 
3. Three months after last vaccine (approximately month 6) toxicity information will be collected from the 
patient’s primary oncologist which will include recent notes and labs. Recent imaging reports may be 
collected if available. 
4. Nine months after the last vaccine (approximately month 12) patients will undergo research blood 
draw of 200 mls for immunologic monitoring. 

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5. Information on patient’s disease status will be obtained biannually from their primary oncologist for up 
to 3 years from enrollment. 
E.  Management of Potential Study Risks 
Although patients have a small chance of experiencing an allergic-type reaction to the vaccine, if a reaction 
were to happen, it would usually occur within one hour of the vaccination.  For this reason, all patients will 
undergo post-vaccination monitoring in the UW CRC which will include observation and monitoring of vital 
signs for a minimum of 60 minutes.  
Patients must have a baseline LVEF measured by MUGA or ECHO equal to or greater than the lower limit 
of normal for the facility within three months of enrollment.  Subsequent LVEF monitoring while on HER2-
targeted monoclonal antibody therapy will be done per standard of care by patient’s oncologist or as 
clinically indicated. 
9. Patient Withdrawal from Study 
The Principal Investigator/Study Physician may deem it appropriate to remove a patient from study 
participation for toxicity (as described in Section 12), recurrent disease, or other reasons.   
Patients who are no longer being vaccinated or provided PSK/Placebo may continue to be followed for 
disease status and/or research blood may be requested to follow immunity.  Whether it was the decision 
of the patient or the Principal Investigator/Study Physician to withdraw participation from the study, it will 
be reported in the annual renewal report to all appropriate agencies.  We will report the reason for the 
withdrawal and whether it was related to the study. 
Patients who have persistent grade 3 or 4 toxicity at time of withdrawal from study treatment will continue 
to be followed until toxicity resolves or returns to baseline for that patient. 
Patients may decide to withdraw from the study at any time and for any reason, at which time all research-
related treatment procedures (vaccine and/or PSK/Placebo) will then cease.   
10. Evaluation and Management of Toxicity 
Toxicity grading will be evaluated according to the CTEP CTCAE v. 4.0 common terminology and the 
DSMP (Appendix C).  DLT will be defined as Grade 2 autoimmune reactions and any incidence of Grade 
3 or 4 adverse events (AEs) other than those listed in Table 5. 
For grade 2 treatment-related reactions, patients will be treated with acetaminophen and/or 
diphenhydramine or as clinically indicated per standard of care; and will not receive further vaccinations 
or PSK. 
Table 5: Expected and Allowable Grade 3 Toxicities (CTCAE v4.0) that are not DLT 
Category 
Toxicity/Adverse Event 
Allowable Duration 
General disorders and administration 
Flu-like symptoms 
1 week 
Musculoskeletal and connective tissue 
disorders 
Arthralgia 
1 week 
Myalgia 
1 week 
Investigations 
Lymphocyte count decreased 
2 week 
Hemoglobin decrease 
2 week 
White blood cells decreased 
2 week 
 
Rationale for allowing the above Grade 3 toxicity is based on the following.  Our proposed target 
population (patients with Stage 4 breast cancer who are NED or have stable bone disease) most likely 
have received ≥2 regimens of chemotherapy which can result in long-lasting grade 1 and 2 hematologic 
abnormalities specifically decreased WBC, lymphocyte counts and hemoglobin which are clinically 
insignificant (e.g., do not place patient at significant increased risk and/or require medical intervention).  
We have extensive experience in vaccinating this patient population and it is expected that despite their 
Grade 1 and 2 hematologic abnormalities, these patients will have fairly normal and functional bone 
marrow reserve and not be at increased risk of harm due to transient perturbations in the immune system.  
Additionally, we have now conducted 3 HER2 vaccine studies in over 100 optimally treated patients with 
stage 4 NED and/or stable bone disease on trastuzumab monotherapy and have observed only transient 
Grade 3 hematologic toxicity that did not require medical intervention.  While the incidence of transient 

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Grade 3 hematologic toxicity in our previous vaccine studies is low (<2%) it can occur due to patients 
having low blood counts at baseline.  The combination of trastuzumab and pertuzumab as maintenance 
therapy has also been extensively studied and found to have an acceptable toxicity profile with no 
increased cardiotoxicity.[43]  Additionally, all patients in our proposed target population will be on either 
trastuzumab or combination trastuzumab/pertuzumab; and the majority will be on endocrine and/or 
bisphosphonate therapy.  All of these therapies independent of vaccination and PSK can incur flu-like 
symptoms, myalgias and arthralgias and enhance similar and expected symptoms related to vaccination.  
Lastly, PSK has not been associated with grade 3 flu-like symptoms, arthralgias, myalgias or hematologic 
toxicity (lymphopenia, anemia, or leukopenia). 
We will monitor patients closely for the above Grade 3 toxicities and should they occur will only be allowed 
for a limited duration of 1-2 weeks.  If any of these Grade 3 toxicities exceed the 1-2 week period, they 
will constitute DLT and patient would not receive further treatment. 
If there is a Serious Adverse Event (Appendix C; Section C), that in the opinion of the Independent Medical 
Monitor, may constitute increased risk to other study participants (i.e., risk section of consent may need 
to be amended), un-blinding of treatment assignment for an individual subject may be requested by the 
medical monitor.  The study statistician would provide the code if requested directly by the medical 
monitor.  The study team would remain blinded and would follow the recommendations of the Independent 
Medical Monitor to amend the necessary study documents.  
11. Dose Modifications 
There will not be any modifications to the vaccine, PSK or placebo doses.  There may be allowances for 
the timing of the administration of vaccine to accommodate patient schedules or special circumstances.  
Subsequent vaccinations will be scheduled 28 + 7 days from when the previous vaccine was actually 
administered, not when it should have been administered. 
12. Accrual and Criteria for Premature Study Termination   
Combination immunotherapy with HER2-directed immunotherapy has been shown to be both safe and 
immunogenic.  Additionally, the proposed oral dose of PSK (3000 mg/day) has been demonstrated to be 
safe with and without chemotherapy in large randomized breast cancer studies with the most common 
and expected AEs being diarrhea, nausea, vomiting, anorexia, hives and stomach discomfort (incidence 
of all listed AEs was <1%).[44, 45]  Although we expect minimal toxicity from HER2 vaccination administered 
concomitantly with PSK, criteria for stopping the trial for excessive treatment-related toxicity will be 
instituted as follows: 
The study will be terminated for safety reasons if there is sufficient evidence to suggest that the true 
incidence of Grade 3 or higher toxicity exceeds 40%, the true incidence of Grade 4 toxicity exceeds 20%, 
or both.  The toxicity rates (adverse events) will be evaluated after every 5th enrolled patient has become 
evaluable, and sufficient evidence will be defined as observed rates of toxicity that correspond to 1-sided 
80% confidence intervals with lower limits greater than 40% (Grade 3 or higher) or 20% (Grade 4).  
Operationally, these limits would be achieved if 4/5, 6/10, or 9/15 patients experienced Grade 3 or higher 
toxicity, or if 3/5, 4/10, or 5/15 patients experienced Grade 4 toxicity.  Monitoring and reporting of AEs and 
toxicity rates will be done per the DSMP (Appendix C). 
13. Statistical Considerations 
A. Study statistics 
1. Determination of Sample Size:  
This sample size is chosen to be sufficient for gathering preliminary data on both the safety of PSK 
and its efficacy in enhancing NK cell activity when combined with HER2 vaccination and HER2 
monoclonal antibody therapyin this patient population.  Thirty patients will be enrolled and randomized 
in equal numbers to one of 2 arms (15 patients/arm) unless there is excessive toxicity as defined in 
Section 12.  Up to 5 additional patients may be enrolled per arm to replace subjects who fail to receive 
study treatment to meet the target accrual of 30 subjects. 
2. Randomization: 
Eligible subjects will be enrolled and randomly assigned in equal proportions to one of two treatment 
arms, Arms 1 (Placebo) and 2 (PSK) via a permuted block design.  Following an algorithm developed 
by the study statistician, a randomization table will be generated and provided to IDS pharmacy.  

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Patients, clinicians and study staff will be blinded to the treatment assignment.  The dispensing UW 
IDS pharmacy staff will be un-blinded. 
a. Randomization will take place following completion of the screening evaluations and eligibility 
assessment. 
b. Patient information will be entered into the study database and patients will be assigned a unique 
study identification number. 
c. The study staff will notify the dispensing pharmacy to request the patient’s randomization. 
d. Patients will be randomly assigned per the randomization table described above. 
3. Statistical Analysis of Study Endpoints: 
This is a randomized phase I/II double-blind study designed to examine the safety and immune effects 
of PSK when given as combination HER2-directed immunotherapy in patients with advanced HER2+ 
breast cancer.   
a. Evaluation of safety and systemic toxicity among the different treatment arms.  Clinical and/or 
chemical parameters for all study patients will be evaluated for potential toxicity at baseline, before 
each vaccine, and 1 month after the third vaccine.  Toxicity grading will be evaluated per CTEP 
CTCAE 4.0 and monitoring of AEs will be done per FDA and NCI guidelines.  The type and grade 
of toxicities noted during treatment will be summarized.  AEs will be tabulated per CTCAE 4.0 
system organ class and descriptive statistics will be used to summarize toxicity profiles for the 
PSK and placebo groups.  Stopping rules based on toxicity are described in Section 12. 
b. Evaluation of the effect of PSK on NK cell functional activity.  Our previous in vitro studies on PSK 
have shown that PSK can enhance NK cell function.  It significantly induced IFN-γ production and 
CD107a expression in NK cells.  After overnight stimulation with 100ug/mL PSK, the percentage 
of NK cells that express CD107a is 1.8+/-1.3% in control group and 6.1+/-3.8% in PSK group (n=7, 
p=0.01).  Therefore we chose CD107a expression as the primary marker of NK cell activation.  
However we do not have direct information on the ability of PSK to activate NK cells after in vivo 
administration.  Based on a recent Phase 1 clinical trial of Trametes versicolor (PSK) in women 
with breast cancer conducted by Standish et al, NK cell activity increased to approximately 2-fold 
of baseline after 4 weeks of oral administration of PSK.  We hypothesize that oral administration 
of PSK will have a similar effect and augment NK cell activity by 2-fold while NK cell activity in the 
placebo group will be unchanged compared to the baseline (pre-treatment) level.  Under these 
assumptions and the assumption that the standard deviation of change in NK cell activity is 1.6, a 
sample size of 15 will provide 91% power (at the 2-sided significance level of .05) to test the null 
hypothesis that the change (post-treatment vs. pre-treatment) in CD107a expression is the same 
in the PSK group as it is in the control group.  These assumptions imply a true effect size of 2/1.6 
= 1.25. 
c. Assessment of the association between changes in pro-inflammatory serum cytokine and/or 
chemokines and treatment with PSK.  Serial blood draws will be done at baseline before the initial 
dose of PSK (time 0) and 30 min (+15 min), 4 hours (+/-15 min), and 24 hours (+/-30 min) after 
the initial dose of PSK to assess serum cytokines/chemokines (i.e., IL-6, IFN-γ, and TNF-α) via 
Luminex analysis.  Serum cytokine/chemokine levels at different timepoints post-PSK will be 
compared to baseline levels using linear regression or linear mixed models.  A global treatment 
group comparison will be made for all post-baseline measurements, for the rate of change in 
cytokine/chemokine levels, and for the 24 hour timepoint.  
d. Assessment of the association between development of intermolecular epitope spreading and 
treatment with PSK.  Preliminary data show that HER2 Th1 vaccines given with trastuzumab elicit 
or augment tumor-specific immunity including epitope spreading.  We have identified a panel of 
breast cancer associated antigens that could provide coverage to approximately 61% of all breast 
cancer patients as an immunogenic profile (HER2, IGFBP-2, TOPO-IIα, p53, and PRAME).  We 
will evaluate PBMC from patients pre- and post-treatment for the development of T cell immunity 
to these antigens by ELISPOT (indicating epitope spreading).  We hypothesize that PSK will boost 
circulating IFN-γ secreting tumor-specific Th1 CD4+ in treated patients.  The development of a 
positive antigen-specific T cell immune response will be defined as a precursor frequency more 
robust than 1:20,000 antigen-specific PBMCs.  In patients without a pre-existent immune 

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response, the acquisition of a positive immune response after treatment begins will constitute 
augmentation.  In patients with pre-existent immune response to the tumor antigens, augmentation 
will have occurred if the final response to tumor antigen is >2 times the baseline level.  Significant 
differences in the levels of immunity to HER2 will be compared between groups using a two-tailed 
T test.  Intramolecular epitope spreading will be defined as generation of significant immunity to 
the HER2 ECD as the patients will be receiving an ICD vaccine.   
The degree of intermolecular epitope spreading in each arm may be summarized as the proportion 
of the 5 antigens that exhibit T cell augmentation as defined above.  If, on average, 2/5 antigens 
are expected to demonstrate epitope spreading in Arm 1 (vaccine and HER2-targeted monoclonal 
antibody therapy alone) and the expected rate is 4/5 in Arm 2 (PSK arm), a generalized linear 
model of binomial proportions with 15 patients per group (two-sided test with α=0.05) will have 
99% power to detect a difference in proportion of antigens that exhibit epitope spreading, and 77% 
power to detect a rate of 4/5 compared to 3/5.  We will also assess the correlation of reduction in 
TGF-β level with the extent of epitope spreading, where each is quantified as a continuous linear 
variable.  Linear regression will be used to assess this correlation, and all patients (both PSK and 
non-PSK patients) will be used for this analysis. 
e. Assessment of the association between changes in TGF-β levels and treatment with PSK.  As 
previously described, PSK may directly inhibit TGF-β signaling resulting in decreased TGF-β 
production and enhanced NK cell activity and increased IFN-γ secretion.  Additionally, our previous 
study data demonstrated vaccine-induced HER2 specific IFN-γ T cell responses to be associated 
with modest reductions in serum TGF-β levels.  We hypothesize that addition of PSK to HER2 
vaccination and HER2-targeted monoclonal antibody therapy will result in increased NK cell-
induced IFN-γ production; and a greater decrease from baseline to post-vaccination serum TGF-
β levels compared to HER2 vaccination and HER2-targeted monoclonal antibody therapy alone.   
In our previous vaccine trial, decrease in serum TGF-β levels among patients who receive HER2 
ICD vaccination and trastuzumab alone was modest.  Specifically, the mean decrease in TGF-β 
(pre-vaccine vs. post-vaccine) was 294 pg/ml (10% reduction) with a standard deviation of roughly 
1000 pg/ml in 23 vaccinated patients.  The average pre-vaccine value among these patients was 
just over 2900 pg/ml.  If we assume that the true decrease in TGF-β level among patients who 
receive PSK is 1450 pg/ml (50%), a sample size of 30 provides 89% power to conclude that 
change in TGF-β is different for groups with and without PSK, using two-sided tests with α=0.05.  
Actual power should be greater, since the statistical analysis will use the pre-test TGF-β as a 
covariate in a linear regression model with transformations performed as needed to satisfy 
regression assumptions.[46]  Combined analysis of multiple post-baseline immune measurements 
(using generalized estimating equations) may also increase precision. 
f. Evaluation of PFS and OS.  PFS and OS (time of first vaccine to time of first event) will be 
compared between treatment arm cohorts.  This study was not designed to be sufficiently powered 
for any particular hypothesis testing, since time-to-event analyses generally require larger sample 
sizes than simple two-way comparisons.  However, large differences, if observed between the 
different vaccine treatment groups will be noted and described. 
B. Projected gender and ethnic distribution 
The ethnic and gender distribution chart below (Table 6) reflects estimates of race and gender of the 
population to be included in this study.  These estimates are based on the following general statistics of 
the population in Washington State based on recent 2010 US Census data.  The population pool from 
which patients will be drawn is 83.8% Caucasian, 7.0% Asian American, 3.9% African American, 1.8% 
American Indian or Alaska Native and 0.5% Native Hawaiian or Other Pacific Islander.  Although men with 
breast cancer are not excluded from this study, less than 1% of all breast cancers occur in men.   
 
Table 6.  Targeted/Planned Enrollment: Number of Subjects = 30 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
3 
0 
3 

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Not Hispanic or Latino 
26 
1 
27 
Ethnic Category: Total of All Subjects * 
29 
1 
30 
Racial Categories 
 
American Indian/Alaska Native 
1 
0 
1 
Asian 
2 
0 
2 
Native Hawaiian or Other Pacific Islander  
0 
0 
0 
Black or African American  
1 
0 
1 
White 
25 
1 
26 
Racial Categories: Total of All Subjects * 
29 
1 
30 
14. Administrative considerations 
A. Institutional review board 
In accordance with federal regulations, an Institutional Review Board that complies with the regulations in 
21 CFR 56 must review and approve this protocol and the informed consent form prior to initiation of the 
study. 
B. Consent 
The P.I., study physician or their designated physician extender must explain verbally and in writing the 
nature and duration of the study and possible consequences of the treatment.  Patients should also be 
informed that they may withdraw from the study at any time and for any reason without jeopardizing their 
future treatment.  At the initiation of eligibility screening and prior to enrollment, the investigators or their 
associates will obtain permission from the patient to access medical records for research purposes.  
Additionally, patients will remain blinded until publication of study manuscript.   
C. Reporting adverse events  
Adverse events will be reported using the NCI’s CTEP CTCAE v 4.0.  A copy of the CTCAE v 4.0 can be 
downloaded from the CTEP homepage (http://ctep.info.nih.gov).  Guidelines for adverse event reporting 
to the FDA and the NCI are described in the DSMP, Appendix C. 
All serious adverse events are communicated to the Principal Investigator, IRB, FDA, NCCAM, CRC, and 
Independent Medical Monitor.  A status report including accrual, adverse events and death information 
will be reviewed by the Principal Investigator and the FHCRC – Cancer Consortium Protocol Data 
Monitoring Committee (PDMC) annually.  In addition, the study will be monitored by the CTSO according 
to the FHCRC – Cancer Consortium monitoring plan. 
D. Confidentiality of patient records 
All eligible patients will be assigned a unique study ID number that will not contain any personally 
identifying information, such as name, initials, medical record number, social security number, etc.  The 
study ID number will be used on all specimens, research study records and associated documents 
collected in the research record.  To maintain confidentiality, we protect the link between the patients’ 
personal identifying information and study ID number by limiting who have access to the patients’ chart 
documentation.  Only specified clinical research staff has access to the data, which remains locked at all 
times when not in use. 
All hard copy research records collected on potential and enrolled patients are stored in a locked filing 
cabinet that is accessed only by approved clinical research staff.  This staff which includes the Principal 
Investigator, study physician(s), and designated clinical research staff are the only ones with access to the 
link between patients’ personal identifying information and their assigned UPN codes. 
Protections and security of electronic clinical data it is being performed by the UW Medicine Information 
Technology (IT) Services.  The UW Medicine IT security policy is to protect UW Medicine information and 
information systems; and ensure compliance with UW policy, state and federal regulations.   
E. Study Team Roles and Responsibilities 
Dr. Gwin, the Principal Investigator will be responsible for the oversight of the research.  Dr. Gwin  is 
responsible for ensuring that all information and documentation related to the conduct and safety of the 
study is disseminated to the proper agencies in the proper timeframe.  Dr. Gwin will delegate tasks to 
qualified research staff for this study as needed. 

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Research physicians/physician extenders will be responsible for conducting consent conferences and 
eligibility determination at the initial visit.  They will perform monthly assessments prior to vaccination.  In 
addition, they will be available to answer any patient questions.  
Research Nurses/Coordinators will be responsible for initial screening and scheduling patient visits, that 
data is entered into the database in a timely manner, so as to have real time data to review and report any 
safety concerns trends.  They will also be responsible for maintaining regulatory documentation to the 
various agencies involved with this research and ensure that all research team members are following the 
protocol and all regulations.   

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41. Metzger-Filho, O., E.P. Winer, et al., Pertuzumab: optimizing HER2 blockade. Clin Cancer Res, 2013. 
19(20): p. 5552-6. 
42. Kumler, I., M.K. Tuxen, et al., A systematic review of dual targeting in HER2-positive breast cancer. 
Cancer Treat Rev, 2014. 40(2): p. 259-70. 
43. Swain, S.M., S.B. Kim, et al., Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastatic 
breast cancer (CLEOPATRA study): overall survival results from a randomised, double-blind, 
placebo-controlled, phase 3 study. Lancet Oncol, 2013. 14(6): p. 461-71. 
44. Toi, M., T. Hattori, et al., Randomized adjuvant trial to evaluate the addition of tamoxifen and PSK to 
chemotherapy in patients with primary breast cancer. 5-Year results from the Nishi-Nippon Group 
of the Adjuvant Chemoendocrine Therapy for Breast Cancer Organization. Cancer, 1992. 70(10): 
p. 2475-83. 
45. Morimoto, T., M. Ogawa, et al., Postoperative adjuvant randomised trial comparing chemoendocrine 
therapy, chemotherapy and immunotherapy for patients with stage II breast cancer: 5-year results 

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from the Nishinihon Cooperative Study Group of Adjuvant Chemoendocrine Therapy for Breast 
Cancer (ACETBC) of Japan. Eur J Cancer, 1996. 32A(2): p. 235-42. 
46. Laird, N., Further Comparative Analyses of Pretest-Posttest Research Designs. American Statistician, 
1983. 37(4): p. 329-330. 
 
 
 

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Appendix A 
Zubrod Performance Scale 
 
Performance status will be assessed according to the current ECOG performance scale* 
Grade 
Description 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light 
or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities. Up and 
about more than 50% of waking hours 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair 
5 
Dead 
 
Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., McFadden, E.T., Carbone, 
P.P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin 
Oncol 5:649-655, 1982. 
*The ECOG Performance Status is in the public domain therefore available for public use.  
To duplicate the scale, please cite the reference above and credit the Eastern Cooperative 
Oncology Group, Robert Comis M.D., Group Chair. 
 

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Appendix B 
Schedule of Events 
 
 
Visit Time Point 
Procedures 
Vaccine #1: Day 1 
(First Visit) 
• 
Consent 
• 
Medical history, physical examination,  symptom assessment and 
medication/supplement changes 
• 
Clinical Labs:  CBC and CMP 
• Vital signs and weight 
• Urine pregnancy test if applicable 
• Research Labs: 200 mls for immunologic monitoring  
• Tetanus-diphtheria (Td) immunization 
• HER2 Vaccination 
• Administration of total daily dose of PSK or placebo then 10 ml blood 
draw 30 minutes and 4 hours post-administration for cytokine kinetics 
Vaccine #1: Day 2 
• Research Labs: 10 mls for cytokine kinetics at CRC then take Day 2 
dose of PSK or placebo as instructed 
• Take home 30 day supply plus 7 additional days  of PSK or placebo 
Vaccine #1: Day 2 until Vaccine #2 visit 
• PSK or placebo daily as instructed and complete daily tea diary 
Vaccine #2: Day 1 
• Interim medical history, physical examination, vital signs and weight 
• Symptom/toxicity assessment and medication/supplement changes 
• Review tea diary 
• 
Clinical Labs:  CBC and CMP 
• Research Labs: 40 mls for immunologic monitoring 
• Urine pregnancy test if applicable 
• HER2 Vaccination 
• Take home 30 day supply plus 7 additional days  of PSK or placebo 
Vaccine #2: Day 1 until Vaccine #3 visit 
• PSK or placebo daily as instructed and complete daily tea diary 
Vaccine #3: Day 1 
• Interim medical history, physical examination, vital signs and weight 
• Symptom/toxicity assessment and medication/supplement changes 
• Review tea diary 
• 
Clinical Labs: CBC and CMP 
• Research Labs: 40 mls for immunologic monitoring 
• Urine pregnancy test if applicable 
• HER2 Vaccination 
• Take home of 30 day supply plus 7 additional days  of PSK or placebo 
Vaccine #3: Day 1 until Month 4 visit 
• PSK or placebo daily as instructed and complete daily tea diary  
Month 4  
(1 month after the last vaccine) 
• Interim medical history, physical examination, vital signs and weight 
• Symptom/toxicity assessment and medication/supplement changes 
• Review tea diary 
• 
Clinical Labs:  CBC and CMP 
• Research blood: 200 mls 
• Take home of 30 day supply of PSK or placebo 
Month 4: Day 1 until Month 5 visit 
• PSK or placebo daily as instructed and complete daily tea diary  
Month 5 
(2 months after the last vaccine) 
• Research Labs: 40 mls for immunologic monitoring  
• Send tea diary to study nurse 
Month 6  
(3 months after the last vaccine) 
• Collection of recent oncologist notes and lab results 
• Collection of recent imaging reports if available 
Month 12 (9 months after last vaccine) 
• Research blood: 200 mls 
• Collection of physician notes including most recent lab results and 
imaging reports 
May be done up to 2 weeks prior to initial vaccine visit 
Clinical Labs:  Complete blood count (CBC) includes differential/platelets; and Comprehensive Metabolic Panel (CMP) includes 
serum electrolytes, creatinine, BUN, AST, ALT, alkaline phosphatase, total bilirubin 

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Appendix C 
Data Safety Monitoring Plan 
A. Purpose 
To ensure that the Tumor Vaccine Group (TVG) follows NIH/NCI/CTEP/FDA guidelines with respect to: 
(1) accurate assessment and timely reporting of adverse drug reactions associated with investigational 
drugs, (2) adherence to protocol, and (3) accurate reporting of data.  The DSMP will be approved by Fred 
Hutchinson Cancer Research Center – Cancer Consortium IRB (FHCRC – CC IRB) in Seattle, 
Washington. 
The Principal Investigator (P.I.), for this project, is responsible for every aspect of the design, delegating 
responsibility/authority, study conduct and final analysis of the protocol.  Regulations defining the 
responsibilities for assessment and reporting of adverse events (AE), serious AE (SAE), and unexpected 
AEs are defined by the Code of Federal Regulations:  21 CFR 312.32 and Common Terminology Criteria 
for Adverse Events (CTCAE) Version 4.0 published by the Cancer Therapy Evaluation Program (CTEP), 
a division of the NCI/NIH. A matrix of reporting requirements and schedules is at the CTEP web-site at 
http://ctep.infi.nih/gov. 
This clinical study will rely upon the monitoring of the trial by the P.I. in conjunction with a Research Nurse, 
a Statistician, an Independent Medical Monitor, and an Independent Study Monitor assigned by the 
Clinical Trials Support Office (CTSO) of the University of Washington (UW)/Fred Hutchinson Cancer 
Research Center (FHCRC) Cancer Consortium.   
The Medical Monitor, Robert Montgomery, M.D. will meet openly with the P.I. and/or their designee, 
Statistician, Research Nurse, Research MD and/or Research Coordinator(s) prior to patients being 
recruited in order to orient committee participants to the study. The Medical Monitor will then discuss the 
study every 6 months throughout the study treatment period with the P.I., Research MD(s), Research 
Nurse(s), Research Coordinator(s), and/or other related clinical research staff. The Medical Monitor will 
review patient recruitment and retention, adherence to protocol, follow-up, data quality, and participant 
risk versus benefit. 
 
B. Objectives 
1. To ensure that the Principal Investigator and clinical research staff follow federal and institutional 
regulatory guidelines with respect to timely reporting of adverse reactions associated with 
investigational drugs.  
2. To define classification of adverse drug reactions as expected or unexpected. 
3. To define classification of adverse drug reactions as serious or non-serious. 
4. To ensure compliance and accuracy of documentation of adverse drug reactions reportable to: (1) 
FHCRC – CC IRB, (2) FDA, (3) UW Clinical Research Center, (4) Independent Medical Monitor and 
(5) NIH - NCCAM. 
 
C. Adverse event reporting policy and procedures 
1. Evaluation of adverse events:  
AEs are graded on a scale of 1-5 and attribution is assigned, using the CTCAE v4 common 
terminology. Patients are monitored at: (1) eligibility, (2) at each vaccination visit, and (3) at Month 4, 
for the development of end organ damage by assessing AEs with serum chemistries, liver function 
studies, complete blood counts and physical exams.  Information pertaining to toxicity is recorded into 
a database AE Log.   
2. Definitions of adverse events:  
Adverse Event - any unfavorable and unintended sign (including abnormal laboratory finding*), 
symptom or disease temporally associated with the use of a medical treatment or procedure 
regardless of whether it is considered related to the medical treatment or procedure.  
 
  

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a. An adverse event may include:  
• 
an exacerbation of a pre-existing illness  
• 
an increase in frequency or intensity of a pre-existing episodic event or condition  
• 
a condition detected or diagnosed after study drug administration  
• 
continuously persistent disease or symptoms that were present at baseline and worsen 
following the start of the study  
• 
*An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
o Results in discontinuation from the study; 
o Is associated with clinical signs or symptoms;  
o Requires treatment or any other therapeutic intervention; 
o Is associated with death or another serious adverse event, including hospitalization; or 
o Is judged by the Investigator to be of significant clinical impact. 
 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 
b. An adverse event does not include:  
• 
medical or surgical procedures (e.g., surgery, endoscopy, tooth extraction, or transfusion); 
however, the condition that leads to the procedure may be an adverse event  
• 
pre-existing diseases, conditions, or laboratory abnormalities present or detected at the start 
of the study that do not worsen  
• 
hospitalizations or procedures that are done for elective purposes not related to an untoward 
medical 
occurrence 
(e.g., 
hospitalizations 
for 
cosmetic 
or 
elective 
surgery 
or 
social/convenience admissions)  
• 
the disease being studied or signs/symptoms associated with the disease unless more severe 
than expected for the patient's condition  
• 
overdose of study drug without any clinical signs or symptoms 
c. Expected Adverse Event - an event that may be reasonably anticipated to occur as a result of the 
study procedure and is described in the Investigators Brochure and/or consent form. 
d. Unexpected Adverse Event - an AE that is not described in the investigator brochure and/or 
consent form and is unanticipated.  An event that might have been anticipated but is more serious 
than expected or occurs more frequently than expected, would be considered an unexpected AE. 
e. Life-threatening Adverse Event - the patient was at substantial risk of dying at the time of the AE 
or it is suspected that the use or continued use of the product would have resulted in the patient’s 
death. 
f. Serious Adverse Event - grade 4 or 5 toxicity or any AE occurring at any dose that results in any 
of the following outcomes: death, a life threatening AE, inpatient hospitalization or prolongation of 
existing hospitalization due to the AE, a persistent or significant disability/incapacity or a congenital 
anomaly/birth defect that is unexpected and unrelated.  
g. Hospitalizations that do not meet these criteria are:  
• 
reasons described in the protocol, e.g., drug administration, protocol-required testing  
• 
social reason in the absence of an AE  
• 
surgery or procedure planned prior to entry into the trial 
Note: Medical and scientific judgment should be exercised in deciding whether expedited reporting is 
appropriate in other situations; for example, important medical events may not be immediately life-

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threatening or result in death or hospitalization but may jeopardize the patient or may require 
intervention to prevent one of the outcomes listed in the definition above.  Any AE is considered a 
serious AE if it is associated with clinical signs or symptoms judged by the investigator to have a 
significant clinical impact.  
3. Adverse event score  
a. Grade 1 = mild adverse event 
b. Grade 2 = moderate adverse event 
c. Grade 3 = severe adverse event 
d. Grade 4 = life-threatening or disabling adverse event 
e. Grade 5 = death related to adverse event. 
 
4. Attribution of adverse event 
a. 5 (definite): the adverse event is clearly related to the investigational agent   
b. 4 (probable): the adverse event is likely related to the investigational agent 
c. 3 (possible): the adverse event may be related to the investigational agent 
d. 2 (unlikely): the adverse event is doubtfully related to the investigational agent 
e. 1 (unrelated): the adverse event is clearly NOT related to the investigational agent 
5.  Adverse event reporting 
This procedure is outlined below by regulatory agency (Table 1). 
Fred Hutchinson Cancer Research Center: Cancer Consortium IRB - We will follow the current AE 
reporting policy of the FHCRC – CC IRB. 
FDA (for trials using an Investigational New Drug (IND) – Adverse events will be reported per 21 CFR 
312.32 by telephone or facsimile transmission (using MedWatch Form 3500A) as soon as possible but no 
later than 7 calendar days after initial receipt of the information concerning the event.  All unexpected, 
serious adverse events are reported in writing (using MedWatch Form 3500A) to the FDA (Center for 
Biologics) within 15 calendar days after initial receipt of the information concerning the event.  Yearly 
written progress reports to the FDA will summarize expected or non-serious unexpected AEs. 
NCCAM – Serious adverse events will be reported to NCCAM Program Officer within 24 hours of FDA 
notification.  A copy of the documents submitted to the FDA will also be submitted to NCCAM within 7-15 
calendar days. Submit written summaries of all reviews conducted by the monitoring group to the 
responsible NCCAM Program Officer within 30 days of reviews or meetings. If reviews are frequent, 
quarterly reports are sufficient. 
University of Washington Clinical Research Center (UW CRC) – The CRC will receive copies of the 
documents submitted to the FHCRC – CC IRB within 7-10 calendar days. 
Independent Medical Monitor - At a minimum, the medical monitor should comment on the outcomes of 
serious adverse event(s) and relationship of the AE to the vaccine or prepare an unbiased written report 
of the event. They should also indicate whether they agree with the details of the report provided by the 
study investigator. 
Table 1.  Adverse event reporting 
 
Expected 
Expected 
Unexpected 
Unexpected 
 
Non-Serious 
Serious 
Non-Serious 
Serious (including 
grade 4 & 5 toxicity) 
 
Attribution* 
(Possible, 
Probable, Definite) 
Attribution * 
(Possible, 
Probable, Definite) 
Attribution * 
(Possible, 
Probable, Definite) 
Attribution * 
(Possible, Probable, 
Definite) 
FHCRC – IRB 
Continuation 
Review Report 
Continuation 
Review Report 
Continuation 
Review Report 
FHCRC Adverse 
Event Expedited 

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Expected 
Expected 
Unexpected 
Unexpected 
 
Non-Serious 
Serious 
Non-Serious 
Serious (including 
grade 4 & 5 toxicity) 
 
Attribution* 
(Possible, 
Probable, Definite) 
Attribution * 
(Possible, 
Probable, Definite) 
Attribution * 
(Possible, 
Probable, Definite) 
Attribution * 
(Possible, Probable, 
Definite) 
Reporting Form – 
ASAP but within 7 
days 
FDA 
Annual Progress 
Report 
Annual Progress 
Report 
Annual Progress 
Report 
FDA Form 3500A – 
ASAP but within 7 
days 
NCCAM 
Annual Review 
Annual Review 
Annual Review 
Notify Program 
Officer within 24 
hours of FDA 
notification 
 
FDA Form 3500A – 
ASAP but within 7-
15 days 
UW Clinical 
Research 
Center (CRC) 
Copy of 
Continuation 
Review Report 
Copy of 
Continuation 
Review Report 
Copy of 
Continuation 
Review Report 
Copy of FHCRC 
Adverse Event 
Expedited Reporting 
Form 
Medical 
Monitor 
Bi-annual Meeting 
(twice a year) 
Bi-annual Meeting 
(twice a year) 
Bi-annual Meeting 
(twice a year) 
Copy of FHCRC 
Adverse Event 
Expedited Reporting 
Form 
 
 
6.  Procedure for reporting serious adverse events: 
a. Identify the classification/attribution of the AE as defined above using CTCAE v4. 
b. After appropriate medical intervention has been instituted, the P.I. or his/her designee will be 
notified within 24 hours. 
c. File appropriate reports immediately by phone/fax with appropriate agencies, as described in Table 
1. 
d. Notify the patient’s primary physician or referring physician within a medically appropriate 
timeframe, depending on the classification of the adverse event. 
e. Submit written reports to appropriate agencies. 
f. Document the AE in the patient’s research file, using a progress note to describe the event and 
treatment, if appropriate.   
g. File copies of all forms/correspondence relating to the AE in the patient’s research file. 
 
D. Clinical trials monitoring operational procedures (Table 2) 
1. Clinical data documentation 
a. Internal study monitoring: 
All patients actively enrolled in the study are seen monthly during the treatment period and one month 
following the final vaccine administration. Clinical labs are evaluated at baseline, before each 
vaccination and 1 month after the 3rd vaccine for the development of toxicity (adverse events) related 
to the vaccine.  Any clinically significant abnormal lab values may be faxed to the patient’s physician.  
The Research Physician, physician extender or P.I. sees each study participant at each visit and the 
following evaluations are completed: toxicity evaluation, physical assessment and AE summary (these 

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evaluations are part of the source documentation that is filled out at each visit).  Grade 3 and 4 non-
serious and expected AEs will be reviewed with the P.I. regularly at clinical meetings.  All other AEs 
will be reported to the P.I. at the time they become known and reported as outlined above in Section 
C. 
Each patient research file is audited for completeness.  Audits may be conducted by a designated 
clinical research staff member on an ongoing basis and the results may be presented clinical meetings.  
Patient’s research files are audited for completeness, legibility, and accuracy.  Clinical research staff 
members will review all patient research files for patient recruitment and retention, protocol adherence, 
follow-up, data quality, and participant risk versus benefit.   
Additionally, correspondence with primary physicians and patients is maintained in the patient 
research file.  Copies of email communications and of verbal communications are maintained.  
b. Biannual review with the independent Medical Monitor: 
The Medical Monitor will meet/or conference call with the P.I. or designee, and other members of the 
clinical team, approximately every 6 months. All patients are reviewed for AEs. Conduct of the study 
is reviewed for any practice changes and documentation of proper notification of changes as 
appropriate to the IRB and FDA. 
c. Biannual study audit:  
An independent Study Monitor will be assigned by the CTSO of the FHCRC/UW Division of Oncology 
and an independent study audit may be performed two times a year (approximately every 6 months).  
This audit will be conducted on a select percentage of enrolled patients.  Patient research files will be 
formally audited for completeness, accuracy and compliance to protocol. In addition, a certain 
percentage of all computer data entry will also be reviewed.  In addition to the routine visit of the UW 
IDS pharmacy, the monitor will also confirm the correct randomized product (PSK/Placebo) was 
dispensed to the patient. 
2. Data validity/integrity 
Systems to insure data integrity have been put into place to provide multiple checks to data entry.  Patient 
eligibility is initially reviewed by the Research Coordinator/Research Nurse at screening, by the Research 
MD prior to enrollment in select cases, approved by the P.I. before enrollment, and biannually by the 
Independent Study Monitor of a certain percentage of charts.  Source documents are initially reviewed by 
the Research Nurse and/or any clinical research staff member involved with recruitment for completeness.   
Clinical laboratory monitoring data is reviewed by a qualified clinician (Research MD, physician extender, 
Research Nurse and/or P.I.) and any abnormalities are assessed.  All patients’ records are kept in the 
patient’s research file and reviewed at biannual monitoring visits.  All toxicity scoring is done by a study 
clinician.  
Data is taken from the patient’s source documentation, the patient’s research file, and entered into a 
database which links data to patient by a Unique Patient Identifier and is accessible by password code 
only.  Data entry is made by the Research Coordinator/designated clinical research staff member and is 
verified at end of study by the Research Nurse/designated clinical research staff member reviewing source 
documents and case report forms; and reconciling them to the study database.  A percentage of all data 
entry is reviewed by a clinical research team member and the Independent Study Monitor.  At the time of 
study reports or publications, print outs of all data from the database are reviewed by the P.I. 
Table 2.  Operational procedures  
Study Event 
Specific Action 
Performed By 
Screen 
Screening of interested 
patients 
Research Coordinator/physician 
extender/Research Nurse/other 
designated clinical research staff 
member assigned by PI 
Eligibility 
Initial review w/interested 
patient 
Research Coordinator/physician 
extender/Research Nurse/other 
designated clinical research staff 
member assigned by PI 

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Study Event 
Specific Action 
Performed By 
 
Obtain source documents to 
determine eligibility 
Research Coordinator/Research 
Nurse/other designated clinical 
research staff member assigned by PI 
 
2nd review of eligibility with final 
approval from the PI 
Research MD/P.I. 
 
Biannual review  
Independent Study and Medical 
Monitors 
Consent 
Obtain Informed Consent 
P.I/ Research MD/physician extender 
Monthly Visits 
Blood draw/ Urine Collection  
Research Nurse (including nurses from 
CRC)/physician extender 
 
Physical assessment/toxicity 
evaluation  
P.I/ Research MD/physician extender 
 
Vaccination 
Research Nurse (including nurses from 
CRC)/physician extender 
 
Monitor for immediate reactions Research Nurse (including nurses from 
CRC), Study Physician 
Toxicity Monitoring 
Monitoring of AEs with patient 
at each visit 
Review of non serious and 
grade 2 expected AEs regularly 
Research MD/P.I./physician extender 
 
P.I. 
 
Biannual review of 
documentation of AEs 
Independent Study  and Medical 
Monitors 
Report of serious 
adverse events 
See sections B and C of DSMP 
Research Nurse/P.I./Physician 
Extender/Research MD/ Research 
Coordinator 
Immunologic Monitoring- 
Research data 
Performed at specified 
timepoints for each patient 
Immunologic Monitoring Lab 
Technologist(s) 
 
Review of immunologic data 
PI /Research MD 
 
Computer entry of immunologic 
data  
Research Coordinator/physician 
extender/other designated clinical 
research staff member assigned by PI 
Study review with P.I. 
 
 
 
 
 
 
Regularly scheduled meeting to 
report study status/evaluate 
eligibility, patient recruitment 
and retention, protocol 
adherence, and AE 
evaluations. Daily on an as 
needed basis for all 
unexpected grade 3-5 AEs 
P.I./Research MD/Physician 
Extender/Research Nurse/Research 
Coordinator 
Study Review by 
Medical Monitor 
Biannual evaluation of patient 
recruitment and retention, 
protocol adherence, AEs and 
participant risk versus benefit. 
Medical Monitor, P.I., Research MD, 
Research Nurse, and Research 
Coordinator 
Twice yearly chart audits Biannual evaluation of patient 
recruitment and retention, 
protocol adherence, AEs and 
participant risk versus benefit. 
Validation of computer data 
entry from the source 
document. 
Independent Study Monitor w/ wrap up 
meeting with the P.I./Study 
physician/physician extender/Research 
Nurse/Research Coordinator(s)  
Assurance of Data 
Accuracy and Integrity 
Validation of computer data 
entry for clinical data 
Research Nurse and Research 
Coordinator(s) 

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Study Event 
Specific Action 
Performed By 
 
Validation of data entry for 
immunologic monitoring-
research data 
Independent Study and Medical 
Monitors/P.I. 
Annual reports to:  
 
 
IRB 
 
 
PI with Research  
Coordinator(s)/Research Nurse 
FDA 
 
PI with Research  
Coordinator(s)/Research Nurse 
CRC 
 
PI with Research  
Coordinator(s)/Research Nurse 
NCCAM 
 
PI with Research  
Coordinator(s)/Research Nurse