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 whi le 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 us ing 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 d evelopment 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 tu mor-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 7866/135 08/23/2021 Page 25 of 39 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 receiv ing 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 demonstrat e 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 a nd 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 ser um TGF- β levels compared to HER 2 vaccination and HER2 -targeted monoclonal antibody therapy alone. In our previous vaccine trial, decrease in ser um 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 t o satisfy regression assumptions .[46] Combined analys is 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 t o time of first event) will be compared between treatment arm cohorts. Thi s 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 7866/135 08/23/2021 Page 26 of 39 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 t reatment. 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 FH CRC – 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 ha ve 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 Medic ine IT security policy is to protect UW Medicine information and information systems ; and ensure compliance with UW policy , state and federal regulations. E.