title testing the ability of
untitled h2

Immune MicroEnvironment (TIME) of high risk DCIS
CC\# 16704 IND NUMBER: 130487  
| Version Number:         | 6.1                                                                                                                 |
|-------------------------|---------------------------------------------------------------------------------------------------------------------|
| Version Date:           | October 23, 2018                                                                                                    |
| Study Drug:             | Pembrolizumab                                                                                                       |
| Study Site:             | University of California, San Francisco                                                                             |
| Principal Investigator: | Laura Esserman, MD, MBA Director, UCSF Carol Franc Buck Breast Care Center Professor of Surgery and Radiology, UCSF |  
Co-Investigators: Jasmine Wong, MD  
  
Michael Campbell, PhD Cheryl Ewing, MD Michael Alvarado, MD Amy Jo Chien, MD Rita Mukhtar, MD  
| Statistician: Study Coordinator: Merck Supplied Agent:   | Pembrolizumab   |
|----------------------------------------------------------|-----------------|  
  
Proprietary and Confidential The information in this document is considered privileged and confidential, and may not be disclosed to others except to the extent necessary to obtain Institutional Review Board approval and informed consent, or as required by Federal and State laws. Persons to whom this information is disclosed should be informed that this information is privileged and confidential and that it should not be further disclosed.
title testing the ability of
protocol signature page

Protocol No.: 16704 **Version Date:** 23OCT2016  
1. I agree to follow this protocol version as approved by the UCSF Protocol Review Committee (PRC), Institutional Review Board (IRB), and Data Safety Monitoring Committee (DSMC).
2. I will conduct the study in accordance with applicable IRB requirements, Federal regulations, and state and local laws to maintain the protection of the rights and welfare of study participants.  
3. I certify that I, and the study staff, have received the requisite training to conduct this research protocol.  
4. I have read and understand the information in the Investigators' Brochure (or Manufacturer's Brochure) regarding the risks and potential benefits. I agree to conduct the protocol in accordance with Good Clinical Practices (ICH-GCP), the applicable ethical principles, the Statement of Investigator (Form FDA 1572), and with local regulatory requirements. In accordance with the FDA Modernization Act, I will ensure the registration of the trial on the www.clinicaltrials.gov website.
5. I agree to maintain adequate and accurate records in accordance with IRB policies, Federal, state and local laws and regulations.
title testing the ability of
table of contents

| Protocol Signature Page                                                                                                                            |                                                                                                                                                   |
|----------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------|
| 1.0                                                                                                                                                | TRIAL SUMMARY ................................................................................................................................7   |
| 2.0                                                                                                                                                | TRIAL DESIGN .                                                                                                                                    |
| Trial Design Summary ..........................................................................................................7                   |                                                                                                                                                   |
| 2.1                                                                                                                                                |                                                                                                                                                   |
| 2.2                                                                                                                                                | Trial Diagram ..................................................................................................................................8 |
| 3.0                                                                                                                                                | OBJECTIVES .                                                                                                                                      |
| 3.1 Primary objectives (dose escalation phase) ......................................................................9                             |                                                                                                                                                   |
| 3.2  Primary objective (dose expansion phase) .........................................................................9                           |                                                                                                                                                   |
| 3.3  Exploratory objectives ...................................................................................................................9   |                                                                                                                                                   |
| 4.0                                                                                                                                                | BACKGROUND & RATIONALE ........................................................................................10                                 |
| Background ..............................................................................................................................10        |                                                                                                                                                   |
| 4.1                                                                                                                                                |                                                                                                                                                   |
| 4.1.1                                                                                                                                              | Pharmaceutical and Therapeutic Background ...........................................................10                                           |
| Preclinical and Clinical Trial Data....................................................................................11                          |                                                                                                                                                   |
| 4.1.2                                                                                                                                              |                                                                                                                                                   |
| 4.2                                                                                                                                                | RATIONALE                                                                                                                                         |
| 4.2.1                                                                                                                                              | Rationale for the Trial and Selected Subject Population ...........................................11                                             |
| 4.2.2                                                                                                                                              | Rationale for Dose Selection/Regimen/Modification...............................................13                                                |
| 4.3                                                                                                                                                | Correlative Studies ....................................................................................................................14        |
| 5.0                                                                                                                                                | Methodology ................................................................................................................................15    |
| 5.1 Entry Criteria .............................................................................................................................15 |                                                                                                                                                   |
| 5.1.1                                                                                                                                              | Diagnosis/Condition for Entry into the Trial                                                                                                      |
| 5.1.2                                                                                                                                              | Subject Inclusion Criteria .......................................................................................................15              |
| __                                                                                                                                                 |                                                                                                                                                   |  
C\# 16704 5.1.3    Subject Exclusion Criteria ..........................................................................................17  
| Study design and investigational treatment .................................................................18      |                                                                                                                                                   |
|---------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------|
| 5.2                                                                                                                 | Definition of Dose-Limiting Toxicities ...........................................................................19                              |
| 5.2.1                                                                                                               |                                                                                                                                                   |
| 5.2.2                                                                                                               | Dose Selection/Modification ...............................................................................................20                     |
| 5.2.3                                                                                                               | Timing of Dose Administration                                                                                                                     |
| 5.2.4                                                                                                               | Trial Blinding/Masking..........................................................................................................25                |
| 5.3                                                                                                                 | Treatment Allocation .........................................................................................................25                  |
| 5.4                                                                                                                 | Stratification                                                                                                                                    |
| 5.5                                                                                                                 | Concomitant Medications/Vaccinations (allowed & prohibited)....................25                                                                 |
| Acceptable Concomitant Medications ..............................................................................25 |                                                                                                                                                   |
| 5.5.1                                                                                                               | Prohibited Concomitant Medications ............................................................................26                                 |
| 5.5.2                                                                                                               |                                                                                                                                                   |
| 5.6                                                                                                                 | Rescue Medications & Supportive Care....................................................................26                                        |
| 5.6.1                                                                                                               | Supportive Care Guidelines ................................................................................................26                     |
| 5.7                                                                                                                 | Diet/Activity/Other Considerations .........................................................................30                                    |
| 5.7.1                                                                                                               | Diet.................................................................................................................................30           |
| 5.7.2                                                                                                               | Contraception                                                                                                                                     |
| 5.7.3                                                                                                               | Use in Pregnancy ................................................................................................................30               |
| 5.7.4                                                                                                               | Use in Nursing Women                                                                                                                              |
| Subject Withdrawal/Discontinuation Criteria.......................................................31                |                                                                                                                                                   |
| 5.8                                                                                                                 |                                                                                                                                                   |
| 5.9                                                                                                                 | Subject Replacement Strategy                                                                                                                      |
| 5.10   Clinical Criteria for Early Trial Termination ..........................................................32   |                                                                                                                                                   |
| 6.0                                                                                                                 | TRIAL FLOW CHART ..............................................................................................................................33 |
| 6.1                                                                                                                 | Study Flow Chart........................................................................................................................33        |
| 7.0                                                                                                                 | TRIAL PROCEDURES ...........................................................................................................34                    |
| __                                                                                                                  |                                                                                                                                                   |
| Pilot: Intralesional Injection Pembrolizumab                                                                        | Page 4 of 102                                                                                                                                     |  
Page 4 of 102  
| 7.1   | Trial Procedures 34                                    |
|-------|--------------------------------------------------------|
| 7.1.1 | Administrative Procedures 35                           |
| 7.1.2 | General Informed Consent 35                            |
| 7.1.3 | Clinical Procedures/Assessments 37                     |
| 7.1.4 | Laboratory Procedures/Assessments 38                   |
| 7.1.5 | Other Procedures 40                                    |
| 7.1.6 | Visit Requirements 40                                  |
| 7.2   | Assessing and Recording Adverse Events 41              |
| 7.2.1 | Definitions of Adverse Events 41                       |
| 7.2.2 | Recording of an Adverse Event 43                       |
| 7.2.3 | Follow-up of Adverse Events 44                         |
| 7.2.4 | Adverse Events Monitoring 44                           |
| 7.2.5 | Expedited Reporting 45                                 |
| 7.2.6 | Evaluating Adverse Events 50                           |
| 7.2.7 | Sponsor Responsibility for Reporting Adverse Events 53 |
| 8.0   | STATISTICAL ANALYSIS PLAN 53                           |  
8.1 Analysis of primary endpoints (dose escalation cohort) .........................................53  
| 9.0                                          | LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL  SUPPLIES 54   |
|----------------------------------------------|--------------------------------------------------------------------|
| 9.1                                          | Investigational Product 54                                         |
| 9.2                                          | Packaging and Labeling Information 54                              |
| 9.3                                          | Clinical Supplies Disclosure 54                                    |
| 9.4                                          | Storage and Handling Requirements 54                               |
| 9.5                                          | Returns and Reconciliation 55                                      |
| 10.0                                         | ADMINISTRATIVE AND REGULATORY DETAILS 55                           |
| Pilot: Intralesional Injection Pembrolizumab | Page 5 of 102                                                      |  
| 10.1                                                                       | Pre-study Documentation 55                         |
|----------------------------------------------------------------------------|----------------------------------------------------|
| 10.2                                                                       | Institutional Review Board Approval 55             |
| 10.3                                                                       | Informed Consent 56                                |
| 10.4                                                                       | Changes in the Protocol 56                         |
| 10.5                                                                       | Handling and Documentation of Clinical Supplies 56 |
| 10.6                                                                       | Case Report Forms (CRFs) 56                        |
| 10.7                                                                       | Oversight and Monitoring Plan 57                   |
| 10.8                                                                       | Regulatory Documentation 57                        |
| 10.9                                                                       | Protection of Human Subjects 58                    |
| 10.9.1 Protection from Unnecessary Harm 58 10.9.2 Protection of Privacy 58 |                                                    |  
| 11.0   | References 59   |
|--------|-----------------|  
12.0 APPENDICES............................................................................................................61 12.1 ECOG Performance Status and Contraceptive Guidance ................................61 12.2 Common Terminology Criteria for Adverse Events V4.0 (CTCAE) ...............65 12.3 UCSF Policy/Procedure for required regulatory documents for single site and multicenter investigator-initiated oncology clinical trials .................................66 12.4 Data and Safety Monitoring Plan for a Phase 1/2 Institutional Study .............71 12.5 EVENT OF CLINICAL INTEREST GUIDANCE DOCUMENT (V3.0)........73
title testing the ability of
1.0 trial summary 1.0 trial

| Abbreviated Title           | Testing the ability of Pembrolizumab to alter the tumor  immune microenvironment of high risk DCIS                                                  |
|-----------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------|
| Trial Phase                 | Single agent pilot study                                                                                                                            |
| Clinical Indication         | High risk DCIS                                                                                                                                      |
| Trial Type                  | Pilot                                                                                                                                               |
| Type of control             | Untreated, proceed to surgical treatment at least 2 weeks after  diagnosis                                                                          |
| Route of administration     | Intralesional                                                                                                                                       |
| Trial Blinding              | None                                                                                                                                                |
| Treatment Groups            | Controls; Intralesional injection of agent                                                                                                          |
| Number of trial subjects    | 3 dose cohorts using a 3+3 cohort dose escalation design (9  patients) 30 in dose expansion group (10 controls, 20  treatment) total of 39 patients |
| Estimated enrollment period | 42 months - June 2016 - December 2020                                                                                                               |
| Estimated duration of trial | 48 months - June 2020 target date for submission of  publication                                                                                    |
| Duration of Participation   | 9 weeks (dose escalation), 5-9 weeks or 8-15 weeks (dose  expansion)                                                                                |
title testing the ability of
2.0 trial design 2.1 trial

We propose a pilot study to investigate the change in the immune microenvironment of high risk ductal carcinoma in situ (DCIS) after short term exposure to pembrolizumab. This study will include 3 dose cohorts using a 3+3 cohort dose escalation design (see figure 1) followed by a 4th cohort at the maximum tolerated dose. Unless a dose limiting toxicity (DLT), defined any grade 3 or 4 toxicity, is observed requiring expansion of a cohort or a subject withdraws, 3 subjects will be enrolled into each cohort in the dose escalation phase. Subjects, upon diagnosis with high risk DCIS, will be offered 2 doses of pembrolizumab injected intralesionally (IL) 3 weeks apart (+/- 1 week) with surgery 3 weeks (+/- 2 weeks) after the 2nd dose. The subject will then undergo the surgical treatment as determined by the surgeon and the subject (partial mastectomy or  
mastectomy). The primary objective of this phase of the study will be safety and feasibility of intralesional injection of pembrolizumab. The maximum tolerated dose will be used in the expansion phase. The expansion cohort will have a target enrollment of 30 subjects enrolled to either the control group or the treatment group. The treatment group will consist of 20 subjects who agree to receive the treatment. The control group will consist of 10 eligible subjects who decline treatment and agree to tissue collection and to the use of tissue for research purposes. The control group will proceed to surgery alone within a 4 month timeframe following the diagnosis of high risk DCIS. The treatment group will receive 4 doses of intralesional pembrolizumab 3 weeks apart (+/- 1 week) prior to surgery. All subjects in the expansion cohort will also undergo a baseline MRI at diagnosis and undergo a 2nd MRI prior to surgery. Baseline and pre-surgical MRI
images will be evaluated for changes in tumor volume.  
| Cohort                                                 | Pembrolizumab Dose   | Number of Subjects   |
|--------------------------------------------------------|----------------------|----------------------|
| 1                                                      | 2 mg                 | 3-6                  |
| 2                                                      | 4 mg                 | 3-6                  |
| 3                                                      | 8 mg                 | 3-6                  |
| 4                                                      | 8 mg                 | 30*                  |
| * 10 patient control group, 20 patient treatment group |                      |                      |
title testing the ability of
3.0 objectives 3.1 primary objectives

3.1a) To determine the maximum tolerated dose (MTD), and recommended dose for subsequent expansion cohort, of intralesionally administered pembrolizumab in patients with ductal carcinoma in situ (DCIS) of the breast. 3.1b) To define the dose-limiting toxicities (DLTs), tolerability, and feasibility of intralesional administration of pembrolizumab in patients with DCIS.
title testing the ability of
3.2 primary objective (dose expansion

3.2a) To determine the response rate to intralesional pembrolizumab in patients with DCIS, as measured by an increase (baseline vs. post treatment) in intralesional CD8+ T
cells, compared to untreated controls.
title testing the ability of
3.3 exploratory objectives

3.3a) To determine whether intralesional pembrolizumab decreases tumor volume on MRI imaging. 3.3b) To determine the extent of cell death within the DCIS lesions (pre- vs. post-therapy) using a cleaved caspase 3 IHC assay. 3.3c) To characterize changes in the immune landscape of DCIS following intralesional administration of pembrolizumab. 3.3d). To characterize changes in peripheral blood-based immune biomarkers.
title testing the ability of
4.0 background & rationale 4.1

Pembrolizumab is a potent humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting its interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor blocking activity for PD-1. Pembrolizumab has an acceptable preclinical safety profile and is in clinical development as an intravenous (IV) immunotherapy for advanced malignancies.  
Keytruda® (pembrolizumab) is indicated for the treatment of patients across a number of indications because of its mechanism of action to bind the PD-1 receptor on the T cell. For more details on specific indications refer to the Investigator brochure.
title testing the ability of
4.1.1 pharmaceutical and therapeutic background

The importance of intact immune surveillance in controlling outgrowth of neoplastic transformation has been known for decades. Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in various malignancies. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to correlate with improved prognosis and long-term survival in many solid tumors. The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T-cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member related to CD28 and CTLA-4 which has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2). The structure of murine PD-1 has been resolved.  
PD-1 and family members are type I transmembrane glycoproteins containing an Ig Variable-type
(V-type) domain responsible for ligand binding and a cytoplasmic tail which is responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3ζ, PKCθ and ZAP70 which are involved in the CD3 T-cell signaling cascade. The mechanism by which PD-1 down modulates T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an overlapping set of signaling proteins. PD-1 was shown to be expressed on activated lymphocytes including peripheral CD4+ and CD8+ T-cells, B-cells, T regs and Natural Killer cells. Expression has also been shown during thymic development on CD4-CD8- (double negative) T-cells as well as subsets of macrophages and dendritic cells. The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic tissues as well  
as in various tumors. Both ligands are type I transmembrane receptors containing both IgV- and IgC-like domains in the extracellular region and contain short cytoplasmic regions with no known signaling motifs. Binding of either PD-1 ligand to PD-1 inhibits T-cell activation triggered through the T-cell receptor. PD-L1 is expressed at low levels on various non-hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed on antigenpresenting cells found in lymphoid tissue or chronic inflammatory environments. PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues. Although healthy organs express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant levels of this T-cell inhibitor. PD-1 has been suggested to regulate tumor-specific T-cell expansion in subjects with melanoma (MEL). This suggests that the PD-1/PD-L1 pathway plays a critical role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention.  
Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PDL1 and PD-L2. KeytrudaTM (pembrolizumab) has recently been approved in the United Stated for the treatment of patients with unresectable or metastatic melanoma and disease progression following ipilumumab and, if BRAF V600 mutation positive, a BRAF inhibitor.
title testing the ability of
4.2 rationale 4.2.1 rationale for

Ductal carcinoma in situ (DCIS) of the breast is a premalignant condition. Although DCIS is treated as an obligate precursor of invasive ductal carcinoma, the rate and latency of progression from DCIS to invasive breast cancer (IBC) in the absence of treatment are unknown. DCIS itself is not a lethal condition, but women with DCIS are at higher risk of developing subsequent local or metastatic IBC over a time period of 1-20 years depending on DCIS subtype.  
DCIS is not one condition, but rather a spectrum of disease ranging from indolent lesions more akin to markers of increased risk for ER positive disease over 20 years and true precursors of triple negative or Her2 positive cancers that are likely to arise in a one to two year timeframe. The latter likely only comprises about 20% of the DCIS diagnosed. Features of DCIS that are associated with high risk of recurrence include large size (> 5cm), high grade, comedo necrosis, palpable mass, hormone receptor (HR) negativity, and HER2 positivity.  
We selected such cases to investigate the immune microenvironment.1-3 Our studies, as well as those of others, have shown that high grade, HR-negative invasive breast cancers have a greater inflammatory component (significant macrophage and T cell infiltration)
compared with low grade, HR-positive tumors.4-8 Importantly, tumor associated macrophages are associated with early recurrence in HR-negative invasive breast cancer. High grade DCIS are also Page 11 of 102  
characterized by a high proportion of macrophages as measured by CD68 positive cells by immunohistochemistry.9-11 To further characterize the immune microenvironment of DCIS, we recently completed an IHC study of 53 cases of high grade DCIS, enriched for large lesions and history of recurrence, age matched with 65 cases of non-high grade DCIS. Immunohistochemical analyses were performed as single color stains for the following antigens: CD115, FoxP3, Ki-67, and HER2. Two color IHC was performed for the following antigen pairs: CD68/PCNA; CD68/Mac 387; CD8/HLA-DR; CD68/MRC1, and CD4/CD20. Stromal TILs were visually estimated from H&E stained sections as a percentage of total stroma per section. HR status was determined from ER and PR staining results in pathology reports. For each case, 3 hot spots were identified and marked on 10 consecutive sections. Nuance multispectral imaging software was used to image each hot spot. Protocols for automated image analysis were developed using CellProfiler software. Clinical parameters of interest included tumor palpability, recurrence, and Van Nuys Prognostic Index, (VNPI, 12 point scale-margins, age, size, grade). Associations were identified with non-parametric Spearman correlation test. In general, immune infiltrates were correlated with high risk DCIS features (high VNPI, palpability, high grade, comedo necrosis, high proliferation, HER2-positivity, and HR-negativity). Comparing the mean percentages of immune cell populations in cases with a recurrence versus those without, we found no cell types that, by themselves, were associated with outcomes. We then constructed a prognostic model using the RPART function in R. A classification tree was generated using as input 16 immune cell populations along with 8 clinical parameters (grade, VNPI, tumor size, palpable, comedonecrosis, HR status, HER2 status, and DCIS density). Of the 24 input parameters, the best RPART model was built using only 3 immune cell populations:
CD8+HLADR+ cells, CD8+HLADR- cells and CD115+ cells. This model had an accuracy of 87%
(sensitivity=76%; specificity=89%). Importantly, all four cases with metastatic recurrences were correctly predicted.  
The highest risk of recurrence was in cases with low numbers of activated CD8+HLADR+ cells.  
Cases with high CD8+HLADR+ cells, but also high numbers of non-activated CD8+HLADR- cells and high numbers of CD115+ cells were also at a high risk for recurrence. In contrast, cases with high CD8+HLADR+ cells and low CD8+HLADR- cells or high CD8+HLADR+ cells, high CD8+HLADR- cells, and low CD115+ cells were at a low risk for recurrence.  
Using HLA-DR as a marker of T cell activation, we observed that activated CD8+HLA-DR+ T cells were associated with good outcomes whereas non-activated or suppressed CD8+HLA-DR- T
cells were associated with increased risk of recurrence. This suggests that activating the CD8+HLA-DR- T cells, perhaps via checkpoint blockade, could potentially alter disease progression. These high-risk DCIS lesions truly represent an opportunity to prevent cancer. Tools to measure the complexity of the tumor immune microenvironment may provide an early measure of preventative potential. The focus of this proposal is to see if the immune microenvironment can be changed with short term exposure to an immunomodulating agent, pembrolizumab. This setting  
will provide an ideal opportunity to evaluate short term single agent exposure, using diagnostic core biopsies (pre) and surgical resection specimens (post).
title testing the ability of
4.2.2 rationale for dose selection/regimen/modification

An open-label Phase I trial (Protocol 001) is being conducted to evaluate the safety and clinical activity of single agent pembrolizumab. The dose escalation portion of this trial evaluated three dose levels, 1 mg/kg, 3 mg/kg, and 10 mg/kg, administered every 2 weeks (Q2W) in subjects with advanced solid tumors. All three dose levels were well tolerated and no dose-limiting toxicities were observed. This first in human study of pembrolizumab showed evidence of target engagement and objective evidence of tumor size reduction at all dose levels (1 mg/kg, 3 mg/kg and 10 mg/kg Q2W). No MTD has been identified to date. Recent data from other clinical studies within the pembrolizumab program has shown that a lower dose of pembrolizumab and a less frequent schedule may be sufficient for target engagement and clinical activity. A population pharmacokinetic analysis has been performed using serum concentration time data from 476 patients. Within the resulting population PK model, clearance and volume parameters of pembrolizumab were found to be dependent on body weight. The relationship between clearance and body weight, with an allometric exponent of 0.59, is within the range observed for other antibodies and would support both body weight normalized dosing or a fixed dose across all body weights. Pembrolizumab has been found to have a wide therapeutic range based on the melanoma indication. The differences in exposure for a 200 mg fixed dose regimen relative to a 2 mg/kg Q3W body weight based regimen are anticipated to remain well within the established exposure margins of 0.5 - 5.0 for pembrolizumab in the melanoma indication. The exposure margins are based on the notion of similar efficacy and safety in melanoma at 10 mg/kg Q3W vs. the proposed dose regimen of 2 mg/kg Q3W (i.e. 5-fold higher dose and exposure). The population PK evaluation revealed that there was no significant impact of tumor burden on exposure. In addition, exposure was similar between the NSCLC and melanoma indications. Therefore, there are no anticipated changes in exposure between different indication settings.  
The rationale for further exploration of 2 mg/kg and comparable doses of pembrolizumab in solid tumors is based on: 1) similar efficacy and safety of pembrolizumab when dosed at either 2 mg/kg or 10 mg/kg Q3W in melanoma patients, 2) the flat exposure-response relationships of pembrolizumab for both efficacy and safety in the dose ranges of 2 mg/kg Q3W to 10 mg/kg Q3W, 3) the lack of effect of tumor burden or indication on distribution behavior of pembrolizumab (as assessed by the population PK model) and 4) the assumption that the dynamics of pembrolizumab target engagement will not vary meaningfully with tumor type. The choice of the 200 mg Q3W as an appropriate dose for the switch to fixed dosing is based on simulations performed using the population PK model of pembrolizumab showing that the fixed dose of 200 mg every 3 weeks will provide exposures that 1) are optimally consistent with those obtained with the 2 mg/kg dose every 3 weeks, 2) will maintain individual patient exposures in the exposure range established in melanoma as associated with maximal efficacy response and 3) will maintain individual patients exposure in the exposure range established in melanoma that are well tolerated and safe.  
A fixed dose regimen will simplify the dosing regimen to be more convenient for physicians and to reduce potential for dosing errors. A fixed dosing scheme will also reduce complexity in the logistical chain at treatment facilities and reduce wastage. Preclinical studies in mice have demonstrated the efficacy of immunomodulatory antibodies administered intratumorally at 1/100th the standard systemic dose.12 The intratumoral injection reduces systemic toxicities by giving it locally and at a lower dose. In a mouse lymphoma model, intratumoral administration of 1 ug/dose anti-CTLA4 is as effective as systemic administration of 100 ug/dose. There are additional mouse studies that demonstrate the efficacy of low-dose intratumoral injection of other inmunomodulatory antibodies as well (anti-CD40 and antiCD137).12-16 In the dose escalation phase of this study, no systemic adverse events were observed at the highest dose of 8 mg. Therefore, we propose to use this dose in the expansion cohort. Although we observed dramatic increases in CD8+ T cell infiltrates in the majority of patients post therapy, this was not accompanied by a reduction in the extent of DCIS in these patients. This was likely due to the short time interval between the initiation of treatment and surgical excision. We therefore propose to extend the time between initiation of therapy and surgery, and add additional injections of pembrolizumab during this time. We hypothesize that this will give the immune response more time to act and result in the reduction or elimination of the DCIS lesions.
title testing the ability of
4.3 correlative studies 4.3.1 characterization

H&E stained sections will be obtained from core biopsies (pre-treatment) and surgical specimens (post-treatment). Tumor infiltrating lymphocyte (TIL) counts will be performed using an automated counting algorithm in QuPath. Stromal TIL, intra-lesional TIL, and total TIL counts will be compared in pre and post samples. Formalin-fixed paraffin embedded (FFPE) tissues from core biopsies (pre-treatment) and surgical specimens (post-treatment) will be obtained from all enrolled patients. Multiplex immunofluorescence staining for panels of immune markers will be performed using Opal staining kits (Perkin Elmer). A multispectral imaging platform (Vectra, Perkin Elmer) will be used to analyze stained sections for various populations of infiltrating immune cells. Analysis of samples from control patients (initial core biopsy vs. surgical specimen) will be used to determine what effects (if any) the biopsy procedure has on the local immune microenvironment. Analysis of samples from pembrolizumab treated patients (initial core biopsy vs. surgical specimen) will be used to determine what effects (if any) pembrolizumab treatment has on the local immune microenvironment. In addition, we will use an IHC assay for apoptosis (cleaved caspase 3 staining) to determine if the increase in CD8+ cytotoxic T cells is associated with increased cell death within the DCIS lesions.
title testing the ability of
4.3.2 analysis of peripheral blood

From complete blood count data we will calculate the following leukocyte ratios as potential biomarkers of systemic immune activity:
Page 14 of 102  
a. neutrophil-to-lymphocyte ratio; NLR b. platelet-to-lymphocyte ratio; PLR c. monocyte-to-lymphocyte ratio; MLR d. neutrophil-to-monocyte ratio; NMR e. eosinophil-to-lymphocyte ratio; ELR f. basophil-to-lymphocyte ratio; BLR
These ratios, as well as absolute cell counts, LDH levels, and CRP levels, will be obtained from pre-treatment, on-treatment, and post-treatment samples. This will yield a dynamic picture of peripheral immunity across the course of therapy. We will run correlation analyses between these peripheral blood immune biomarkers and immune cell populations identified by multiplex immunofluorescence in pre- and post-treatment tissue samples to determine if changes in peripheral blood biomarkers predict changes in the tissue immune microenvironment, and ultimately with changes in the extent of DCIS.
title testing the ability of
5.0 methodology 5.1 entry criteria

The patient must have high risk, biopsy proven DCIS. Patients who were previously enrolled on the dose escalation arm are eligible to enroll on the dose expansion arm.
title testing the ability of
5.1.2 subject inclusion criteria

In order to be eligible for participation in this trial, the subject must:
1. Plan on having surgical treatment for her DCIS.  
2. Have at least 2 of the following high risk features associated with her DCIS - high-grade
(grade II-III), palpable mass, hormone receptor negative (less than 1%), Her2 positive, young age (less than 45 years old), and large size (greater than 5 cm).  
3. Patients with a history of tamoxifen and/or aromatase inhibitor use for treatment or prevention are eligible but should discontinue these medications at least 2 weeks prior to starting this trial.
4. Be willing and able to provide written informed consent/assent for the trial. 5. Be 18 years of age on day of signing informed consent. 6. Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1
(defined in Appendix 1).  
7. Demonstrate adequate organ function as defined in Table 1 Adequate Organ Function Laboratory Values. All screening labs should be performed within 10 days of treatment initiation.  
Table 1. Adequate Organ Function Laboratory Values  
| System                                                                                                                                                                             | Laboratory Value                                                                                                                                                                                                                                                                                  |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Hematological Absolute neutrophil count (ANC)                                                                                                                                      | ≥1,500 /mcL                                                                                                                                                                                                                                                                                       |
| Platelets                                                                                                                                                                          | ≥100,000 / mcL                                                                                                                                                                                                                                                                                    |
| Hemoglobin                                                                                                                                                                         | ≥9 g/dL or ≥5.6 mmol/L without transfusion or EPO dependency  (within 7 days of assessment)                                                                                                                                                                                                       |
| Renal Serum creatinine OR Measured or calculateda  creatinine clearance (GFR can also be used in place of creatinine or CrCl)                                                      | ≤1.5 X upper limit of normal (ULN) OR ≥60 mL/min for subject with creatinine levels > 1.5 X institutional  ULN                                                                                                                                                                                    |
| Hepatic Serum total bilirubin                                                                                                                                                      | ≤ 1.5 X ULN OR Direct bilirubin ≤ ULN for subjects with total bilirubin levels > 1.5  ULN                                                                                                                                                                                                         |
| AST (SGOT) and ALT (SGPT)                                                                                                                                                          | ≤ 2.5 X ULN                                                                                                                                                                                                                                                                                       |
| Albumin                                                                                                                                                                            | >2.5 mg/dL                                                                                                                                                                                                                                                                                        |
| Coagulation                                                                                                                                                                        | ≤1.5 X ULN unless subject is receiving anticoagulant therapy as long as PT or PTT is within therapeutic range of intended use of  anticoagulants ≤1.5 X ULN unless subject is receiving anticoagulant therapy as long as PT or PTT is within therapeutic range of intended use of  anticoagulants |
| aCreatinine clearance should be calculated per institutional standard. International Normalized Ratio (INR) or Prothrombin  Time (PT) Activated Partial Thromboplastin Time (aPTT) |                                                                                                                                                                                                                                                                                                   |  
8. A female participant is eligible to participate if she is not pregnant (see Appendix 1.1), not breastfeeding, and at least one of the following conditions applies:
a. Not a woman of childbearing potential (WOCBP) as defined in Appendix 1.1 OR
b. A WOCBP who agrees to follow the contraceptive guidance in Appendix 1.1 during the treatment period and for at least 90 days corresponding to time needed to eliminate any study treatment plus 30 days (a menstruation cycle) after the last dose of study treatment.  
9. A male participant must agree to use a contraception as detailed in Appendix 1.1 of this protocol during the treatment period and for at least 90 days corresponding to time needed to eliminate any study treatment plus an additional 120 days (a spermatogenesis cycle) after the last dose of study treatment and refrain from donating sperm during this period.
title testing the ability of
5.1.3 subject exclusion criteria

The subject must be excluded from participating in the trial if the subject:  
1. Is currently participating and receiving study therapy or has participated in a study of an investigational agent and received study therapy or used an investigational device within 4 weeks of the first dose of treatment.  
2. Has a diagnosis of immunodeficiency or is receiving systemic steroid therapy or any other form of immunosuppressive therapy within 7 days prior to the first dose of trial treatment.
3. Is not interested in surgical treatment for her DCIS. 4. Has invasive breast cancer. This does not include microinvasion. 5. Has a known history of active TB (Bacillus Tuberculosis). 6. Hypersensitivity to pembrolizumab or any of its excipients. 7. Has a known additional malignancy that is progressing or requires active treatment.  
Exceptions include basal cell carcinoma of the skin or squamous cell carcinoma of the skin that has undergone potentially curative therapy or in situ cervical cancer.  
8. Has active autoimmune disease that has required systemic treatment in the past 2 years (i.e.  
with use of disease modifying agents, corticosteroids or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic treatment.
9. Has a history of (non-infectious) pneumonitis that required steroids or has current pneumonitis.  
10. Has a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the trial, interfere with the subject's participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator.  
11. Has known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial.  
12. Is pregnant or breastfeeding, or expecting to conceive or father children within the projected duration of the trial, starting with the pre-screening or screening visit through 120 days after the last dose of trial treatment.  
13. Has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies).  
14. Has a known history of Hepatitis B (defined as Hepatitis B surface antigen [HBsAg]
reactive) or known active Hepatitis C virus (defined as HCV RNA [qualitative] is detected) infection. Note: no testing for Hepatitis B and Hepatitis C is required unless mandated by local health authority.  
15. Has received a live vaccine within 30 days prior to the first dose of study drug. Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella/zoster (chicken pox), yellow fever, rabies, Bacillus Calmette–Guérin (BCG), and typhoid vaccine. Seasonal influenza vaccines for injection are generally killed virus vaccines and are allowed; however, intranasal influenza vaccines (eg, FluMist®) are live attenuated vaccines and are not allowed.
title testing the ability of
5.2 study design and investigational

This is a pilot study in patients with DCIS. The primary objective of this trial is to define the MTD and recommended dose of intralesional pembrolizumab. The dose escalation phase of this study followed a standard 3+3 trial design (Table 2). The initial dose of intralesional pembrolizumab (2 mg) were chosen to be 1:100 the standard IV dose (200 mg) based on preclinical studies with immunomodulatory mAbs. Doses were escalated 2-fold as shown in Table 3. Patients enrolled in the expansion cohort will receive intralesional pembrolizumab at the MTD as determined from the escalation phase. If a MTD is not reached, patients in the expansion cohort will receive the highest dose evaluated (8 mg).  
| Number of Patients with DLT  at a Given Dose Level   | Escalation Decision Rule                                                                                                                                                                                                                                                                                                                                                    |
|------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 0 out of 3                                           | Escalate dose to next higher dose level Enter at least 3 more patients at this dose level If 0 of these 3 additional patients experience DLT (1 of 6), proceed to the next  dose level If 1 or more of the 3 additional patients suffer DLT (2 of 6), then dose  escalation is stopped and this dose is declared the maximal administered dose  (highest dose administered) |
| 1 out of 3                                           | Determination of the MTD will continue at the next lowest dose cohort, at  which an additional 3 patients will be added, for a total of 6 (unless that cohort  already has 6 patients)                                                                                                                                                                                      |
| ≥ 2 out of 3                                         | Dose escalation will be stopped                                                                                                                                                                                                                                                                                                                                             |
| Pilot: Intralesional Injection Pembrolizumab         | Page 18 of 102                                                                                                                                                                                                                                                                                                                                                              |  
Table 2. Dose escalation 3+3 trial design.  
| This dose level is declared the maximal administered dose The next lower cohort will be expanded to 6 patients If < 1 experience DLT, this dose is the maximal tolerated dose (MTD)   |                                                                                                                    |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------|
| ≤ 1 out of 6 at highest dose level  below the maximal administered  dose                                                                                                              | This is generally the recommended Phase 2 dose At least 6 patients must be entered at the recommended Phase 2 dose |
title testing the ability of
5.2.1 definition of dose-limiting toxicities

All toxicities will be graded using National Cancer Institute (NCI) CTCAE Version 4.0 (Appendix 12.2). The occurrence of any of the following toxicities during Cycle 1 will be considered a DLT:
Grade 4 non-hematologic toxicity Grade 3 non-hematologic toxicity lasting >3 days despite optimal supportive care Any Grade 3 or 4 non-hematologic laboratory value if:
o Medical intervention is required to treat the patient, or o The abnormality leads to hospitalization, or o The abnormality persists for >1 week Neutropenia that is:
o Grade 3 or 4 (i.e., ANC <1000 per mm3) and associated with fever (oral temperature ≥39ºC) requiring antibiotic therapy o Grade 4 which lasts >7 days or leads to use of therapeutic G-CSF
Grade 4 thrombocytopenia (i.e., platelets <25,000 per mm3) or Grade 3 thrombocytopenia (i.e., platelets <50,000 per mm3) with bleeding
title testing the ability of
table 3. trial treatment dose

| Drug          | Dose/                  | Dose                                  | Route of          | Regimen/      |              |
|---------------|------------------------|---------------------------------------|-------------------|---------------|--------------|
| Potency       | Frequency              | Administration                        | Treatment  Period | Use           |              |
| Pembrolizumab | 2 mg  (1:100 IV  dose) | 2 doses 3  weeks  apart (+/-  1 week) | Intralesional     | Day 1 of each | Experimental |
| injection     | 3 week cycle           |                                       |                   |               |              |  
| Pembrolizumab   | 4 mg  (1:50 IV  dose)   | 2 doses 3  weeks  apart (+/-  1 week)   | Intralesional   | Day 1 of each   | Experimental   |
|-----------------|-------------------------|-----------------------------------------|-----------------|-----------------|----------------|
| injection       | 3 week cycle            |                                         |                 |                 |                |
| Pembrolizumab   | 8 mg  (1:25 IV  dose)   | 2 doses 3  weeks  apart (+/-  1 week)   | Intralesional   | Day 1 of each   | Experimental   |
| injection       | 3 week cycle            |                                         |                 |                 |                |
title testing the ability of
dose expansion

| Drug            | Dose/                      | Dose          |                 |              |
|-----------------|----------------------------|---------------|-----------------|--------------|
| Potency         | Frequenc y                 | Route         | of              |              |
| Administratio n | Regimen/ Treatment  Period | Use           |                 |              |
| Pembrolizumab   | 8 mg (1:25                 | 4 doses 3     |                 |              |
| IV dose)        | weeks  apart (+/-  1 week) | Intralesional | Day 1 of each 3 | Experimental |
| Injection       | week cycle                 |               |                 |              |  
The patient will proceed to the operating room for the operation as determined by the subject and the surgeon (partial mastectomy versus mastectomy) 3 weeks (+/- 2 weeks) after the 4th dose of intralesional pembrolizumab. Trial treatment should begin on the day of treatment selection or as close as possible to the date on which treatment is allocated/assigned.
title testing the ability of
5.2.2 dose selection/modification 5.2.2.1 dose

The rationale for selection of doses to be used in this trial is provided in Section 4.0 - Background and Rationale. Briefly, based on preclinical studies in mice that have demonstrated the efficacy of immunomodulatory antibodies administered intratumorally at 1/100th the standard systemic dose, we chose a starting dose of 2 mg (1:100 the standard i.v. dose of pembrolizumab). Doses will be escalated 2-fold in each subsequent escalation cohort. Details on preparation and administration of pembrolizumab (MK-3475) are provided in the Pharmacy Manual.  
Page 20 of 102
title testing the ability of
5.2.2.2 dose modification and toxicity

AEs associated with pembrolizumab exposure may represent an immunologic etiology. These immune-related AEs (irAEs) may occur shortly after the first dose or several months after the last dose of pembrolizumab treatment and may affect more than on body system simultaneously. Therefore, early recognition and initiation of treatment is critical to reduce complications. Based on existing clinical study data, most irAEs were reversible and could be managed with interruptions of pembrolizumab, administration of corticosteroids and/or other supportive care. For suspected irAEs, ensure adequate evaluation to confirm etiology or exclude other causes. Additional procedures or tests such as bronchoscopy, endoscopy, skin biopsy may be included as part of the evaluation. Based on the severity of irAEs, withhold or permanently discontinue pembrolizumab and administer corticosteroids. Dose modification and toxicity management guidelines for irAEs associated with pembrolizumab are provided in Table 4.  
Table 4. Dose modification and toxicity management guidelines for immune-related AEs associated with pembrolizumab  
| 1.                                   | Corticosteroid taper should be initiated upon AE improving to Grade 1 or less and continue to taper over at  least 4 weeks.                                                                                                                                                                                                                                                |                       |                      |                                                                                                       |    |                                                              |
|--------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------|----------------------|-------------------------------------------------------------------------------------------------------|----|--------------------------------------------------------------|
| 2.                                   | For situations where pembrolizumab has been withheld, pembrolizumab can be resumed after AE has been  reduced to Grade 1 or 0 and corticosteroid has been tapered. Pembrolizumab should be permanently  discontinued if AE does not resolve within 12 weeks of last dose or corticosteroids cannot be reduced to ≤10  mg prednisone or equivalent per day within 12 weeks. |                       |                      |                                                                                                       |    |                                                              |
| 3.                                   | For severe and life-threatening irAEs, IV corticosteroid should be initiated first followed by oral steroid.  Other immunosuppressive treatment should be initiated if irAEs cannot be controlled by corticosteroids.                                                                                                                                                      |                       |                      |                                                                                                       |    |                                                              |
| Immune-related                       | Toxicity grade                                                                                                                                                                                                                                                                                                                                                             |                       |                      |                                                                                                       |    |                                                              |
| AEs                                  | or conditions  (CTCAEv4.0)                                                                                                                                                                                                                                                                                                                                                 | Action taken to       | irAE management with |                                                                                                       |    |                                                              |
| pembrolizumab                        | corticosteroid and/or other  therapies                                                                                                                                                                                                                                                                                                                                     | Monitor and follow-up |                      |                                                                                                       |    |                                                              |
| Pneumonitis                          | Grade 2                                                                                                                                                                                                                                                                                                                                                                    | Withhold              |                     | Administer  corticosteroids (initial  dose of 1-2 mg/kg  prednisone or equivalent)  followed by taper |   | Monitor participants  for signs and symptoms  of pneumonitis |
|                                     | Evaluate participants  with suspected  pneumonitis with  radiographic imaging  and initiate  corticosteroid treatment                                                                                                                                                                                                                                                      |                       |                      |                                                                                                       |    |                                                              |
|                                     | Add prophylactic  antibiotics for  opportunistic infections                                                                                                                                                                                                                                                                                                                |                       |                      |                                                                                                       |    |                                                              |
| Grade 3 or 4, or  recurrent Grade  2 | Permanently  discontinue                                                                                                                                                                                                                                                                                                                                                   |                       |                      |                                                                                                       |    |                                                              |  
General instructions:  
| Version date: 23OCT2018                             | CC# 16704                                                                                                                                                                                                                 |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
|-----------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|----------------------------------------------------------------------------------------------------------|
| Diarrhea / Colitis                                  | Grade 2 or 3                                                                                                                                                                                                              | Withhold                                                                                                                              |                                                                                                         | Monitor participants for  signs and symptoms of  enterocolitis (ie,  diarrhea, abdominal  pain, blood or mucus in  stool with or without  fever) and of bowel  perforation  (ie, peritoneal signs and  ileus). |    |                                                                                                          |
|                                                    | Participants with ≥  Grade 2 diarrhea  suspecting colitis  should consider GI  consultation and  performing endoscopy  to rule out colitis.                                                                               |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
|                                                    | Participants with  diarrhea/colitis should  be advised to drink  liberal quantities of  clear fluids. If  sufficient oral fluid  intake is not feasible,  fluid and electrolytes  should be substituted  via IV infusion. |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
| Grade 2                                             | Withhold                                                                                                                                                                                                                  |                                                                                                                                      | Administer  corticosteroids (initial  dose of 0.5- 1 mg/kg  prednisone or equivalent)  followed by taper |                                                                                                                                                                                                                |    |                                                                                                          |
|                                                    | Administer  corticosteroids (initial  dose of 1-2 mg/kg  prednisone or equivalent)  followed by taper                                                                                                                     |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
| Grade 4                                             | Permanently  discontinue                                                                                                                                                                                                  |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
| AST / ALT  elevation or  Increased  bilirubin       |                                                                                                                                                                                                                          | Monitor with liver  function tests (consider  weekly or more  frequently until liver  enzyme value returned  to baseline or is stable |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
| Grade 3 or 4                                        | Permanently                                                                                                                                                                                                               |                                                                                                                                      | Administer                                                                                               |                                                                                                                                                                                                                |    |                                                                                                          |
| discontinue                                         | corticosteroids (initial  dose of 1-2 mg/kg  prednisone or equivalent)  followed by taper                                                                                                                                 |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
| Type 1 diabetes  mellitus (T1DM)  or  Hyperglycemia | Newly onset  T1DM or  Grade 3 or 4  hyperglycemia  associated with  evidence of - cell failure                                                                                                                            | Withhold                                                                                                                              |                                                                                                         | Initiate insulin  replacement therapy for  participants with T1DM                                                                                                                                              |    |                                                                                                          |
|                                                    | Administer antihyperglycemic in  participants with  hyperglycemia                                                                                                                                                                                                                           |                                                                                                                                      | Monitor participants  for hyperglycemia or  other signs and  symptoms of diabetes.                       |                                                                                                                                                                                                                |    |                                                                                                          |
| Hypophysitis                                        | Grade 2                                                                                                                                                                                                                   | Withhold                                                                                                                              |                                                                                                         | Administer  corticosteroids and  initiate hormonal  replacements as  clinically indicated.                                                                                                                     |   | Monitor for signs and  symptoms of  hypophysitis (including  hypopituitarism and  adrenal insufficiency) |
| Grade 3 or 4                                        | Withhold or  permanently  discontinue1                                                                                                                                                                                    |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |
| Pilot: Intralesional Injection Pembrolizumab        | Page 22 of 102                                                                                                                                                                                                            |                                                                                                                                       |                                                                                                          |                                                                                                                                                                                                                |    |                                                                                                          |  
| Version date: 23OCT2018                                                                                                                                                                                                                                                                | CC# 16704                                                                                                                                                           |                          |    |                                                                                                     |    |                                                                               |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------|----|-----------------------------------------------------------------------------------------------------|----|-------------------------------------------------------------------------------|
| Hyperthyroidism                                                                                                                                                                                                                                                                        | Grade 2                                                                                                                                                             | Continue                 |   | Treat with non-selective  beta-blockers (eg,  propranolol) or  thionamides as  appropriate          |   | Monitor for signs and  symptoms of thyroid  disorders.                        |
| Grade 3 or 4                                                                                                                                                                                                                                                                           | Withhold or  permanently  discontinue1                                                                                                                              |                          |    |                                                                                                     |    |                                                                               |
| Hypothyroidism                                                                                                                                                                                                                                                                         | Grade 2-4                                                                                                                                                           | Continue                 |   | Initiate thyroid  replacement hormones  (eg, levothyroxine or  liothyroinine) per  standard of care |   | Monitor for signs and  symptoms of thyroid  disorders.                        |
| Nephritis and                                                                                                                                                                                                                                                                          | Grade 2                                                                                                                                                             | Withhold                 |    |                                                                                                     |    |                                                                               |
| Renal  dysfunction                                                                                                                                                                                                                                                                     | Grade 3 or 4                                                                                                                                                        | Permanently  discontinue |   | Administer  corticosteroids  (prednisone 1-2 mg/kg  or equivalent) followed  by taper.              |   | Monitor changes of  renal function                                            |
| Myocarditis                                                                                                                                                                                                                                                                            | Grade 1 or 2                                                                                                                                                        | Withhold                 |   | Based on severity of AE  administer  corticosteroids                                                |   | Ensure adequate  evaluation to confirm  etiology and/or exclude  other causes |
| Grade 3 or 4                                                                                                                                                                                                                                                                           | Permanently  discontinue                                                                                                                                            |                          |    |                                                                                                     |    |                                                                               |
| All other  immune-related  AEs                                                                                                                                                                                                                                                         | Intolerable/  persistent  Grade 2                                                                                                                                   | Withhold                 |   | Based on type and  severity of AE  administer  corticosteroids                                      |   | Ensure adequate  evaluation to confirm  etiology and/or exclude  other causes |
| Grade 3                                                                                                                                                                                                                                                                                | Withhold or  discontinue  based on the  type of event.  Events that  require  discontinuation  include and not  limited to:  Gullain-Barre  Syndrome,  encephalitis |                          |    |                                                                                                     |    |                                                                               |
| Grade 4 or  recurrent Grade  3                                                                                                                                                                                                                                                         | Permanently  discontinue                                                                                                                                            |                          |    |                                                                                                     |    |                                                                               |
| 1.                                                                                                                                                                                                                                                                                     | Withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating physician.                                                   |                          |    |                                                                                                     |    |                                                                               |
| NOTE:  For participants with Grade 3 or 4 immune-related endocrinopathy where withhold of pembrolizumab is required,  pembrolizumab may be resumed when AE resolves to ≤ Grade 2 and is controlled with hormonal replacement  therapy or achieved metabolic control (in case of T1DM). |                                                                                                                                                                     |                          |    |                                                                                                     |    |                                                                               |  
Dosing interruptions are permitted in the case of medical / surgical events or logistical reasons not related to study therapy (e.g., elective surgery, unrelated medical events, patient vacation, and/or holidays). Subjects should be placed back on study therapy within 3 weeks of the scheduled interruption, unless otherwise discussed with the Sponsor. The reason for interruption should be documented in the patient's study record.
title testing the ability of
5.2.3 timing of dose administration

Trial treatment should be administered on Day 1 of each cycle after all procedures/assessments have been completed as detailed on the Trial Flow Chart (Section 6.0). Trial treatment may be administered up to 7 days before or after the scheduled Day 1 of each cycle due to administrative reasons. All trial treatments will be administered on an outpatient basis.
title testing the ability of
dose escalation cohort

For intralesional injection, concentrated pembrolizumab will be injected into the palpable DCIS or in the vicinity of the non-palpable lesion. Injections will be given on 2 occasions 3 weeks apart (+/- 1 week). The surgeon, in clinic, will use ultrasound imaging (an ultrasound visible clip placed at the time of initial biopsy) to guide the placement of the single injection. If a hydromark clip was not used, we will work with the radiologists to localize the clip and perform the injection.
title testing the ability of
dose expansion cohort

Given satisfactory safety data an additional 20 patients will be treated with intralesional injections and 10 patients will act as the control group. The control group will proceed directly to surgery. In the intralesional treated cohort, concentrated pembrolizumab will be injected into the palpable DCIS or in the vicinity of the non-palpable lesion. Injections will be given on four occasions, 3 weeks apart (+/- 1 week). The surgeon, in clinic, will use ultrasound imaging (an ultrasound visible clip placed at the time of initial biopsy) to guide the placement of the single injection. If a hydromark clip was not used, we will work with the radiologists to localize the clip and perform the injection. The patient will proceed to the operating room for the operation as determined by the subject and the surgeon (partial mastectomy versus mastectomy) 3 weeks (+/- 2 weeks) after the 4th dose of intralesional pembrolizumab.  
The Pharmacy Manual contains specific instructions for the preparation of the Pembrolizumab for injection. A 50 mg lyophilized powder single use vial will be reconstituted with sterile water and then diluted to the correct dose for injection using normal saline. The total injection volume will be 2 milliliters.  
| Dose                | Volume of NS to 1 mL of 25   | Volume of injection   |
|---------------------|------------------------------|-----------------------|
| mg/mL pembrolizumab |                              |                       |
| 2 mg/2 mL           | 24 mL                        | 2.05 mL               |
| 4 mg/2 mL           | 11.5 mL                      | 2.05 mL               |  
Page 24 of 102  
| 8 mg/2mL   | 5.25 mL   | 2.05 mL   |
|------------|-----------|-----------|
title testing the ability of
5.2.4 trial blinding/masking

This is an open-label trial; therefore, the Sponsor, investigator and subject will know the treatment administered. The immune markers will be performed and evaluated by a team that does not know treatment status (blinded to study arm).
title testing the ability of
5.3 treatment allocation

In the dose escalation cohort, target enrollment was 3-6 patients at each dose.  
In the expansion cohort, target enrollment in the expansion study will be 10 patients in the control arm and 20 patients in the treatment arm. Patients who agree to receive treatment will be enrolled to the treatment arm. Patients who are eligible to participate but decline treatment will be enrolled to the control arm. The core biopsies and surgical specimen of control patients will be banked for research.
title testing the ability of
5.4 stratification

Based on the limited number of patients in the study, we will not stratify.
title testing the ability of
5.5 concomitant medications/vaccinations (allowed &

Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed during the ongoing trial. If there is a clinical indication for one of these or other medications or vaccinations specifically prohibited during the trial, discontinuation from trial therapy or vaccination may be required. The investigator should discuss any questions regarding this with the Merck Clinical team. The final decision on any supportive therapy or vaccination rests with the investigator and/or the subject's primary physician.
title testing the ability of
5.5.1 acceptable concomitant medications

All treatments that the investigator considers necessary for a subject's welfare may be administered at the discretion of the investigator in keeping with the community standards of medical care. All concomitant medication will be recorded on the case report form (CRF) including all prescription, over-the-counter (OTC), herbal supplements, and IV medications and fluids. If changes occur during the trial period, documentation of drug dosage, frequency, route, and date may also be included on the CRF. All concomitant medications received within 28 days before the first dose of trial treatment and 30 days after the last dose of trial treatment should be recorded. Concomitant medications administered after 30 days after the last dose of trial treatment should be recorded for SAEs and ECIs as defined in Section 7.2.
title testing the ability of
5.5.2 prohibited concomitant medications

Subjects are prohibited from receiving the following therapies during the Screening and Treatment Phase (including retreatment for post-complete response relapse) of this trial:
Antineoplastic systemic chemotherapy or biological therapy Immunotherapy not specified in this protocol Chemotherapy not specified in this protocol Investigational agents other than pembrolizumab Radiation therapy Live vaccines within 30 days prior to the first dose of trial treatment and while participating in the trial. Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella/zoster, yellow fever, rabies, BCG, and typhoid vaccine.  
Systemic glucocorticoids for any purpose other than to modulate symptoms from an event of clinical interest of suspected immunologic etiology. The use of physiologic doses of corticosteroids may be approved after consultation with the Sponsor.  
Subjects who, in the assessment by the investigator, require the use of any of the aforementioned treatments for clinical management should be removed from the trial. Subjects may receive other medications that the investigator deems to be medically necessary. The Exclusion Criteria describes other medications which are prohibited in this trial. There are no prohibited therapies during the Post-Treatment Follow-up Phase.
title testing the ability of
5.6 rescue medications & supportive

Subjects should receive appropriate supportive care measures as deemed necessary by the treating investigator. Suggested supportive care measures for the management of adverse events with potential immunologic etiology are outlined below and in greater detail in the ECI guidance document. Where appropriate, these guidelines include the use of oral or intravenous treatment with corticosteroids as well as additional anti-inflammatory agents if symptoms do not improve with administration of corticosteroids. Note that several courses of steroid tapering may be necessary as symptoms may worsen when the steroid dose is decreased. For each disorder, attempts should be made to rule out other causes such as metastatic disease or bacterial or viral infection, which might require additional supportive care. The treatment guidelines are intended to be applied when the investigator determines the events to be related to pembrolizumab. Note: if after the evaluation the event is determined not to be related, the investigator is instructed to follow the ECI reporting guidance but does not need to follow the treatment guidance (as outlined in the ECI guidance document). Refer to Section 5.2.1 for dose modification.  
Page 26 of 102  
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or skin photography as part of evaluation of the event. Suggested conditional procedures, as appropriate, can be found in the ECI guidance document.
title testing the ability of
pneumonitis

o For **Grade 2 events**, treat with systemic corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks.  
o For **Grade 3-4 events**, immediately treat with intravenous steroids. Administer additional anti-inflammatory measures, as needed.  
o Add prophylactic antibiotics for opportunistic infections in the case of prolonged steroid administration.
title testing the ability of
diarrhea/colitis

Subjects should be carefully monitored for signs and symptoms of enterocolitis (such as diarrhea, abdominal pain, blood or mucus in stool, with or without fever) and of bowel perforation (such as peritoneal signs and ileus).  
o All subjects who experience diarrhea/colitis should be advised to drink liberal quantities of clear fluids. If sufficient oral fluid intake is not feasible, fluid and electrolytes should be substituted via IV infusion. For Grade 2 or higher diarrhea, consider GI consultation and endoscopy to confirm or rule out colitis.  
o For **Grade 2 diarrhea/colitis** that persists greater than 3 days, administer oral corticosteroids.  
o For **Grade 3 or 4 diarrhea/colitis** that persists > 1 week, treat with intravenous steroids followed by high dose oral steroids.  
o When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks.
title testing the ability of
type 1 diabetes mellitus (if

Insulin replacement therapy is recommended for Type I diabetes mellitus and for Grade 3-4 hyperglycemia associated with metabolic acidosis or ketonuria.  
Evaluate patients with serum glucose and a metabolic panel, urine ketones, glycosylated hemoglobin, and C-peptide.
title testing the ability of
hyperthyroidism or hypothyroidism

Thyroid disorders can occur at any time during treatment. Monitor patients for changes in thyroid function (at the start of treatment, periodically during treatment, and as indicated based on clinical evaluation) and for clinical signs and symptoms of thyroid disorders.  
Page 27 of 102 o **Grade 2** hyperthyroidism events (and **Grade 2-4** hypothyroidism):
In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are suggested as initial therapy.  
In hypothyroidism, thyroid hormone replacement therapy, with levothyroxine or liothyroinine, is indicated per standard of care.  
o **Grade 3-4** hyperthyroidism Treat with an initial dose of IV corticosteroid followed by oral corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks. Replacement of appropriate hormones may be required as the steroid dose is tapered.
title testing the ability of
hepatic

o For **Grade 2** events, monitor liver function tests more frequently until returned to baseline values (consider weekly).  
Treat with IV or oral corticosteroids o For **Grade 3-4** events, treat with intravenous corticosteroids for 24 to 48 hours.  
o When symptoms improve to Grade 1 or less, a steroid taper should be started and continued over no less than 4 weeks.
title testing the ability of
renal failure or nephritis

o For **Grade 2** events, treat with corticosteroids.  
o For **Grade 3-4** events, treat with systemic corticosteroids.  
o When symptoms improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 weeks.  
**Management of Local Reactions**: Signs and symptoms may develop during or shortly after drug injection and generally resolve completely within 24 hours of completion of injection. .  
5 below shows treatment guidelines for subjects who experience a local reaction associated with administration of pembrolizumab.  
| Table 5. Injection Reaction Treatment Guidelines NCI CTCAE Grade Treatment                                                                                                                                                                                                                                                                                                    | Premedication at Subsequent  Dosing                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                             |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 1 Mild reaction; infusion  interruption not  indicated; intervention  not indicated                                                                                                                                                                                                                                                                                     | Increase monitoring of vital signs as medically  indicated until the participant is deemed medically  stable in the opinion of the investigator.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | None                                                                                                                                                                                                                        |
| Grade 2 Requires therapy or  infusion interruption but  responds promptly to  symptomatic treatment  (e.g., antihistamines,  NSAIDs, narcotics, IV  fluids); prophylactic  medications indicated  for ≤24 hrs                                                                                                                                                                 | Stop Infusion. Additional appropriate medical therapy may  include but is not limited to: IV fluids Antihistamines NSAIDs Acetaminophen Narcotics Increase monitoring of vital signs as medically  indicated until the participant is deemed medically  stable in the opinion of the investigator. If symptoms resolve within 1 hour of stopping drug  infusion, the infusion may be restarted at 50% of  the original infusion rate (e.g. from 100 mL/hr to 50  mL/hr). Otherwise dosing will be held until  symptoms resolve and the participant should be  premedicated for the next scheduled dose. Participants who develop Grade 2 toxicity  despite adequate premedication should be  permanently discontinued from further study  drug treatment | Participant may be  premedicated 1.5h (± 30  minutes) prior to infusion of  _____ with: Diphenhydramine 50 mg po  (or equivalent dose of  antihistamine). Acetaminophen 500-1000 mg  po (or equivalent dose of  analgesic). |
| Grades 3 or 4 Grade 3: Prolonged (i.e., not  rapidly responsive to  symptomatic medication  and/or brief interruption  of infusion); recurrence  of symptoms following  initial improvement;  hospitalization indicated  for other clinical  sequelae (e.g., renal  impairment, pulmonary  infiltrates) Grade 4: Life-threatening; pressor  or ventilatory support  indicated | Stop Infusion. Additional appropriate medical therapy may  include but is not limited to: Epinephrine** IV fluids Antihistamines NSAIDs Acetaminophen Narcotics Oxygen Pressors Corticosteroids Increase monitoring of vital signs as medically  indicated until the participant is deemed medically  stable in the opinion of the investigator. Hospitalization may be indicated. **In cases of anaphylaxis, epinephrine should be  used immediately. Participant is permanently discontinued from  further study drug treatment.                                                                                                                                                                                                                       | No subsequent dosing                                                                                                                                                                                                        |
| Appropriate resuscitation equipment should be available at the bedside and a physician readily available during the period of  drug administration. For further information, please refer to the Common Terminology Criteria for Adverse Events v4.0 (CTCAE) at  http://ctep.cancer.govPilot: Intralesional Injection Pembrolizumab Page 29 of 102                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                             |
title testing the ability of
5.7 diet/activity/other considerations 5.7.1 diet

Subjects should maintain a normal diet unless modifications are required to manage an AE such as diarrhea, nausea or vomiting.
title testing the ability of
5.7.2 contraception

Pembrolizumab may have adverse effects on a fetus in utero. Refer to Appendix 12.1.1 for approved methods of contraception. Pembrolizumab may have adverse effects on a fetus in utero. Furthermore, it is not known if pembrolizumab has transient adverse effects on the composition of sperm. Non-pregnant, nonbreast-feeding women may be enrolled if they are willing to use 2 methods of birth control or are considered highly unlikely to conceive. Highly unlikely to conceive is defined as 1) surgically sterilized, or 2) postmenopausal (a woman who is ≥45 years of age and has not had menses for greater than 1 year will be considered postmenopausal), or 3) not heterosexually active for the duration of the study. The two birth control methods can be either two barrier methods or a barrier method plus a hormonal method to prevent pregnancy. Subjects should start using birth control from study Visit 1 throughout the study period up to 120 days after the last dose of study therapy. The following are considered adequate barrier methods of contraception: diaphragm, condom (by the partner), copper intrauterine device, sponge, or spermicide. Appropriate hormonal contraceptives will include any registered and marketed contraceptive agent that contains an estrogen and/or a progestational agent (including oral, subcutaneous, intrauterine, or intramuscular agents). Subjects should be informed that taking the study medication may involve unknown risks to the fetus (unborn baby) if pregnancy were to occur during the study. In order to participate in the study they must adhere to the contraception requirement (described above) for the duration of the study and during the follow-up period defined in section 7.2.5.2-Reporting of Pregnancy and Lactation to the Sponsor and to Merck. If there is any question that a subject will not reliably comply with the requirements for contraception, that subject should not be entered into the study.
title testing the ability of
5.7.3 use in pregnancy

If a subject inadvertently becomes pregnant while on treatment with pembrolizumab, the subject will immediately be removed from the study. The site will contact the subject at least monthly and document the subject's status until the pregnancy has been completed or terminated. The outcome of the pregnancy will be reported to the Sponsor and to Merck without delay and within 24 hours to the Sponsor and within 2 working days to Merck if the outcome is a serious adverse experience (e.g., death, abortion, congenital anomaly, or other disabling or life-threatening complication to the mother or newborn).  
The study investigator will make every effort to obtain permission to follow the outcome of the pregnancy and report the condition of the fetus or newborn to the Sponsor. If a male subject impregnates his female partner the study personnel at the site must be informed immediately and the pregnancy reported to the Sponsor and to Merck and followed as described above and in Section 7.2.5.2.
title testing the ability of
5.7.4 use in nursing women

It is unknown whether pembrolizumab is excreted in human milk. Since many drugs are excreted in human milk, and because of the potential for serious adverse reactions in the nursing infant, subjects who are breast-feeding are not eligible for enrollment.
title testing the ability of
5.8 subject withdrawal/discontinuation criteria

Subjects may withdraw consent at any time for any reason or be dropped from the trial at the discretion of the investigator should any untoward effect occur. In addition, a subject may be withdrawn by the investigator or the Sponsor if enrollment into the trial is inappropriate, the trial plan is violated, or for administrative and/or other safety reasons. Specific details regarding discontinuation or withdrawal are provided in Section 7.1.5 - Other Procedures. A subject must be discontinued from the trial for any of the following reasons:
The subject or legal representative (such as a parent or legal guardian) withdraws consent.  
Unacceptable adverse experiences as described in Section 7.2 Intercurrent illness that prevents further administration of treatment The participant has a medical condition or personal circumstance which, in the opinion of the investigator and/or sponsor, placed the participant at unnecessary risk from continued administration of study treatment.  
The subject has a confirmed positive serum pregnancy test Noncompliance with trial treatment or procedure requirements The subject is lost to follow-up Administrative reasons Recurrent Grade 2 pneumonitis Completion of 35 treatments (approximately 2 years) with pembrolizumab
The End of Treatment and Follow-up visit procedures are listed in Section 6 (Protocol Flow Chart) and Section 7.1.6 (Visit Requirements). After the end of treatment, each subject will be followed  
for 30 days for adverse event monitoring (serious adverse events will be collected for 90 days after the end of treatment as described in Section 7.2).
title testing the ability of
5.9 subject replacement strategy

This is an intent to treat study with a short course of therapy. Our goal is to have 20 evaluable patients (as long as there is no safety contraindication after the dose escalation phase). Patients who withdraw consent or opt to go elsewhere for surgical treatment without enabling us to obtain blocks will be replaced. Enrollment will re-open, and an additional eligible patient will be enrolled until we reach a cohort of 20 evaluable patients.
title testing the ability of
5.10 clinical criteria for early

Early trial termination will be the result of the criteria specified below: 1. Quality or quantity of data recording is inaccurate or incomplete  
2. Poor adherence to protocol and regulatory requirements 3. Incidence or severity of adverse drug reaction in this or other studies indicates a potential health hazard to subjects 4. Plans to modify or discontinue the development of the study drug
In the event of Merck decision to no longer supply study drug, ample notification will be provided so that appropriate adjustments to subject treatment can be made.
6.0 trial flow chart 6.1
5.10 clinical criteria for early

| End of                                                         |                    |                    |                |        |          |             |    |    |
|----------------------------------------------------------------|--------------------|--------------------|----------------|--------|----------|-------------|----|----|
| Trial Period                                                   | Treatment Cycles   | Treatment          | Post-Treatment |        |          |             |    |    |
| Safety Followup/Post-op                                                                | Follow Up          |                    |                |        |          |             |    |    |
| Visito                                                         | Visits             |                    |                |        |          |             |    |    |
| Screenin                                                       | 1 st               | 2 nd               | 3 rd           | 4 th   | Surgical |             |    |    |
| Treatment Cycle/Title                                          | g                  | dose               | dose           | dose   | dose     | treatment   | 8 wks +/-  2 weeks  postsurgery    |    |
| Informed Consenta                                              | x                  |                    |                |        |          |             |    |    |
| Review of eligibility criteriab                                | x                  |                    |                |        |          |             |    |    |
| Demographics and Medical  Historyc                             | x                  |                    |                |        |          |             |    |    |
| Prior and Concomitant Medication  Reviewd                      | x                  | x                  | x              | x      | x        | x           | x  |    |
| Intralesional injection  pembrolizumabe                        | x                  | x                  | x              | x      |          |             |    |    |
| Review Adverse Eventsf                                         | x                  | x                  | x              | x      | x        | x           | x  |    |
| Full Physical Examinationg                                     | x                  |                    |                |        |          |             |    |    |
| Directed Physical Examinationh                                 | x                  | x                  | x              | x      | x        | x           |    |    |
| -28 to 0                                                       | 1-5 wks post       | 1- 3 wks postsurgery                    |                |        |          |             |    |    |
| Scheduling Window                                              | days               | ± 1 wk             | ± 1 wk         | ± 1 wk | ± 1 wk   | second dose |    |    |
| Vital Signs and Weighti                                        | x                  | x                  | x              | x      | x        | x           | x  |    |
| ECOG Performance Statusj                                       | x                  | x                  | x              | x      | x        | x           | x  | x  |
| Core                                                           |                    |                    |                |        |          |             |    |    |
| Tissue Collectionk                                             | biopsy  (archival) | Surgical  specimen |                |        |          |             |    |    |
| Laboratory Proceduresl Pregnancy Test - Urine or Serumm        | x                  | x                  |                |        |          |             |    |    |
| Coagulation                                                    | x                  | x                  | x              |        |          |             |    |    |
| CBC with Differential                                          | x                  | x                  | x              |        |          |             |    |    |
| Comprehensive Serum Chemistry  Panel(BMP and LFTs)             | x                  | x                  | x              |        |          |             |    |    |
| Urinalysis                                                     | x                  | x                  | x              |        |          |             |    |    |
| Thyroid Function                                               | x                  | x                  | x              |        |          |             |    |    |
| Imaging ProcedurenPilot: Intralesional Injection Pembrolizumab | Page 33 of 102     |                    |                |        |          |             |    |    |  
| End of                                                                                                                      |                  |           |                |        |          |             |    |
|-----------------------------------------------------------------------------------------------------------------------------|------------------|-----------|----------------|--------|----------|-------------|----|
| Trial Period                                                                                                                | Treatment Cycles | Treatment | Post-Treatment |        |          |             |    |
| Safety Followup/Post-op                                                                                                                             | Follow Up        |           |                |        |          |             |    |
| Visito                                                                                                                      | Visits           |           |                |        |          |             |    |
| Screenin                                                                                                                    | 1 st             | 2 nd      | 3 rd           | 4 th   | Surgical |             |    |
| Treatment Cycle/Title                                                                                                       | g                | dose      | dose           | dose   | dose     | treatment   | 8 wks +/-  2 weeks  postsurgery    |
| -28 to 0                                                                                                                    | 1-5 wks post     | 1- 3 wks postsurgery           |                |        |          |             |    |
| Scheduling Window                                                                                                           | days             | ± 1 wk    | ± 1 wk         | ± 1 wk | ± 1 wk   | second dose |    |
| MRI scan (dose expansion group  only)                                                                                       | x                | x         |                |        |          |             |    |
| aDuring dose expansion cohort: At the time of enrollment patients will be assigned an enrollment number. Patients and their |                  |           |                |        |          |             |    |  
a**During dose expansion cohort:** At the time of enrollment patients will be assigned an enrollment number. Patients and their corresponding enrollment numbers will be logged and organized in OnCore.  
bPatients with a diagnosis of high risk DCIS will come in to the clinic for a screening visit and identified as a potential candidate for the study during their clinic visit (i.e., eligibility). Eligible subjects, identified in clinic by the treating surgeon, will be assigned a screening number by the study coordinator at the time of identification. Screening numbers will be stored in an encrypted, secure Microsoft Excel file.  
cMedical history includes all active conditions, and any condition diagnosed within the prior 10 years that are considered to be clinically significant by the Investigator.  
dThe investigator or qualified designee will record medication, if any, taken by the subject during the trial. All medications related to reportable SAEs and ECIs should be recorded as defined in Section 7.2.  
e**During dose escalation and patients on the treatment arm during dose expansion:** On Day 1 of each 3 week (+/- 1 week)
cycle. Treatment window is +/- 7 days of scheduled Day 1 administration of each cycle due to administrative reasons.  
fSignificant baseline AEs prior to treatment and surgery will be collected.  
gThe investigator or qualified designee will perform a complete physical exam during the screening period. Clinically significant abnormal findings should be recorded as medical history. A full physical exam should be performed during screening, hThe investigator or qualified designee will perform a directed physical exam as clinically indicated prior to trial treatment administration.  
iVital signs include temperature, pulse, respiratory rate, weight and blood pressure. Height will be measured at screening only. jAt screening, prior to the administration of each dose of trial treatment, and at discontinuation of trial treatment.  
kPatients will receive a core biopsy upon diagnosis as part of standard clinical procedure. At surgery, tissue will be banked and sectioned for immunohistological analysis.  
lRepeat labs not necessary provided baseline lab results within normal range. See Table 6 Laboratory Tests for specific tests mPregnancy test must be negative within 3 days prior to Day 1 except in dose expansion cohort who are enrolled to control arm
(surgical treatment only).  
n**During dose expansion cohort:** Patients will undergo MRI imaging of the breast to evaluate the extent and volume of DCIS.  
Patients will be offered an additional breast MRI pre-surgery. Breast MRI images will be evaluated for changes in tumor volume and tumor immune microenvironment.  
oSafety follow-up visit and post-op visit will typically occur during the 2 week mark.  
p Screening procedures can occur on the same day as C1D1. Vital signs, ECOG performance status, adverse event review, and concomitant medication review from that day will be recorded for both visits.
6.0 trial flow chart 6.1
7.0 trial procedures 7.1 trial

The Trial Flow Chart - Section 6.0 summarizes the trial procedures to be performed at each visit. Individual trial procedures are described in detail below. It may be necessary to perform these procedures at unscheduled time points if deemed clinically necessary by the investigator.  
Furthermore, additional evaluations/testing may be deemed necessary by the Sponsor and/or Merck for reasons related to subject safety. In some cases, such evaluation/testing may be potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may require that additional informed consent be obtained from the subject. In these cases, such evaluations/testing will be performed in accordance with those regulations.
6.0 trial flow chart 6.1
7.1.1 administrative procedures 7.1.1.1 informed

The Investigator must obtain documented consent from each potential subject prior to participating in a clinical trial.
6.0 trial flow chart 6.1
7.1.2 general informed consent

Consent must be documented by the subject's dated signature or by the subject's legally acceptable representative's dated signature on a consent form along with the dated signature of the person conducting the consent discussion. A copy of the signed and dated consent form should be given to the subject before participation in the trial. The initial informed consent form, any subsequent revised written informed consent form and any written information provided to the subject must receive the UCSF IRB's approval/favorable opinion in advance of use. The subject or his/her legally acceptable representative should be informed in a timely manner if new information becomes available that may be relevant to the subject's willingness to continue participation in the trial. The communication of this information will be provided and documented via a revised consent form or addendum to the original consent form that captures the subject's dated signature or by the subject's legally acceptable representative's dated signature.  
Specifics about a trial and the trial population will be added to the consent form template at the protocol level. The informed consent will adhere to UCSF IRB requirements, applicable laws and regulations and Sponsor requirements.
6.0 trial flow chart 6.1
7.1.2.1 inclusion/exclusion criteria

All inclusion and exclusion criteria will be reviewed by the investigator or qualified designee to ensure that the subject qualifies for the trial.
6.0 trial flow chart 6.1
7.1.2.2 medical history

A medical history will be obtained by the investigator or qualified designee. Medical history will include all active conditions, and any condition diagnosed within the prior 10 years that are  
considered to be clinically significant by the Investigator. Details regarding the disease for which the subject has enrolled in this study will be recorded separately and not listed as medical history.
6.0 trial flow chart 6.1
7.1.2.3 prior and concomitant medications

The investigator or qualified designee will review prior medication use, including any protocolspecified washout requirement, and record prior medication taken by the subject within 28 days before starting the trial. Treatment for the disease for which the subject has enrolled in this study will be recorded separately and not listed as a prior medication.
6.0 trial flow chart 6.1
7.1.2.3.2 concomitant medications

The investigator or qualified designee will record medication, if any, taken by the subject during the trial. All medications related to reportable SAEs and ECIs should be recorded as defined in Section 7.2.
6.0 trial flow chart 6.1
7.1.2.4 disease details and treatments

The investigator or qualified designee will obtain prior and current details regarding disease status.
6.0 trial flow chart 6.1
7.1.2.5 assignment of screening number

Eligible subjects, identified in clinic by the treating surgeon, will be assigned a screening number by the study coordinator at the time of identification. Any identifying information associated with this number will be destroyed if the patient does not consent to participate in the study. Screening numbers will be stored in an encrypted, secure Microsoft Excel file.
6.0 trial flow chart 6.1
7.1.2.6 assignment of enrollment number

At the time of treatment selection, patients on study in the expansion cohort will be assigned an enrollment number. Patients and their corresponding enrollment numbers will be logged and organized in OnCore - The Online Collaborative Research Environment. This secure system will keep all patient personal health information secure and standardized.
6.0 trial flow chart 6.1
7.1.2.7 trial compliance (medication/diet/activity/other)

Pembrolizumab will be administered in clinic. Subjects on the treatment arm will be required to be present for treatment. Patients will be advised not to change diet or activity and to remain compliant with home medications.
6.0 trial flow chart 6.1
7.1.3 clinical procedures/assessments 7.1.3.1 adverse

The investigator or qualified designee will assess each subject to evaluate for potential new or worsening AEs as specified in the Trial Flow Chart and more frequently if clinically indicated. Adverse experiences will be graded and recorded throughout the study and during the follow-up period according to NCI CTCAE Version 4.0 (see Section 11.2). Toxicities will be characterized in terms regarding seriousness, causality, toxicity grading, and action taken with regard to trial treatment. For subjects receiving treatment with pembrolizumab all AEs of unknown etiology associated with pembrolizumab exposure should be evaluated to determine if it is possibly an event of clinical interest (ECI) of a potentially immunologic etiology (termed immune-related adverse events, or irAEs); see the separate ECI guidance document in Appendix 4 regarding the identification, evaluation and management of potential irAEs. Please refer to section 7.2 for detailed information regarding the assessment and recording of AEs.
6.0 trial flow chart 6.1
7.1.3.2 full physical exam

The investigator or qualified designee will perform a complete physical exam during the screening period. Clinically significant abnormal findings should be recorded as medical history. A full physical exam should be performed during screening,
6.0 trial flow chart 6.1
7.1.3.3 directed physical exam

For cycles that do not require a full physical exam per the Trial Flow Chart, the investigator or qualified designee will perform a directed physical exam as clinically indicated prior to trial treatment administration.
6.0 trial flow chart 6.1
7.1.3.4 vital signs

The investigator or qualified designee will take vital signs at screening, prior to the administration of each dose of trial treatment and at treatment discontinuation as specified in the Trial Flow Chart (Section 6.0). Vital signs should include temperature, pulse, respiratory rate, weight and blood pressure. Height will be measured at screening only.
6.0 trial flow chart 6.1
7.1.3.5 eastern cooperative oncology group

The investigator or qualified designee will assess ECOG status (see Section 11.1) at screening, prior to the administration of each dose of trial treatment and discontinuation of trial treatment as specified in the Trial Flow Chart.
6.0 trial flow chart 6.1
7.1.3.6 tumor imaging and assessment

Patients will undergo MRI imaging of the breast to evaluate the extent and volume of DCIS pretreatment and pre-surgery. Breast MRI images will be evaluated for changes in tumor volume and tumor immune microenvironment.
6.0 trial flow chart 6.1
7.1.3.7 tumor tissue collection and

Patients will receive a core biopsy upon diagnosis as part of standard clinical procedure. At surgery, tissue will be banked and sectioned for immunohistological analysis. Peripheral blood will be collected from patients prior to each injection of pembrolizumab for standard blood counts and chemistries. An additional EDTA tube will be collected at baseline and prior to surgery from which plasma will be frozen for subsequent assays of cytokines and chemokines and white blood cells will be processed and frozen for subsequent flow cytometry or CyTOF analyses.
6.0 trial flow chart 6.1
7.1.4 laboratory procedures/assessments

Details regarding specific laboratory procedures/assessments to be performed in this trial are provided below Laboratory Safety Evaluations (Hematology, Chemistry and Urinalysis) Laboratory tests for hematology, chemistry, urinalysis, and others are specified in Table 6.  
| Hematology                                                                                                                                                                                                 | Chemistry                                                                                                                                                                                           | Urinalysis                     | Other                                   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------|-----------------------------------------|
| Hematocrit                                                                                                                                                                                                 | Albumin                                                                                                                                                                                             | Blood                          | Serum β-human  chorionic  gonadotropin† |
| Hemoglobin                                                                                                                                                                                                 | Alkaline phosphatase                                                                                                                                                                                | Glucose                        | (β-hCG)†                                |
| Platelet count                                                                                                                                                                                             | Alanine aminotransferase (ALT)                                                                                                                                                                      | Protein                        | PT (INR)                                |
| WBC (total and                                                                                                                                                                                             | Aspartate aminotransferase (AST)                                                                                                                                                                    | Specific gravity               | aPTT                                    |
| differential) Red Blood Cell Count                                                                                                                                                                         | Lactate dehydrogenase (LDH)                                                                                                                                                                         | Microscopic exam (If abnormal) | Total  thriiodothyronin e (T3)          |
| Absolute Neutrophil                                                                                                                                                                                        | Carbon Dioxide ‡                                                                                                                                                                                    | results are noted              | Free thyroxine                          |
| Count                                                                                                                                                                                                      | (T4)                                                                                                                                                                                                |                                |                                         |
| Absolute Lymphocyte                                                                                                                                                                                        | (CO2 or biocarbonate)                                                                                                                                                                               | Urine pregnancy test †         | Thyroid                                 |
| Count                                                                                                                                                                                                      | stimulating  hormone (TSH)                                                                                                                                                                          |                                |                                         |
| Monocyte count                                                                                                                                                                                             | Uric Acid                                                                                                                                                                                           |                                |                                         |
| Platelet count                                                                                                                                                                                             | Calcium                                                                                                                                                                                             |                                |                                         |
| Eosinophil count                                                                                                                                                                                           | Chloride                                                                                                                                                                                            |                                |                                         |
| Basophil count                                                                                                                                                                                             | Glucose Phosphorus Potassium  Sodium Magnesium Total Bilirubin Direct Bilirubin (If total bilirubin  is elevated above the upper limit of  normal) Total protein Blood Urea Nitrogen Creatinine CRP |                                |                                         |
| † Perform on women of childbearing potential only. If urine pregnancy results cannot be confirmed as negative, a  serum pregnancy test will be required.  ‡ If considered standard of care in your region. |                                                                                                                                                                                                     |                                |                                         |  
Table 6. Laboratory Tests
6.0 trial flow chart 6.1
7.1.5 other procedures 7.1.5.1 withdrawal/discontinuation

When a subject discontinues/withdraws prior to trial completion, all applicable activities scheduled for the final trial visit should be performed at the time of discontinuation. Any adverse events which are present at the time of discontinuation/withdrawal should be followed in accordance with the safety requirements outlined in Section 7.2 - Assessing and Recording Adverse Events.
6.0 trial flow chart 6.1
7.1.5.2 blinding/unblinding

This trial is open-label; therefore, the subject, the trial site personnel, the Sponsor and/or designee are not blinded to treatment. Drug identity (name, strength) is included in the label text; random code/disclosure envelopes or lists are not provided.
6.0 trial flow chart 6.1
7.1.6 visit requirements

Visit requirements are outlined in Section 6.0 - Trial Flow Chart. Specific procedure-related details are provided above in Section 7.1 - Trial Procedures.
6.0 trial flow chart 6.1
7.1.6.1 screening 7.1.6.1.1 screening period

We will determine eligibility at initial patient consult based on pathology and imaging results. Patients who choose to participate in the study will be enrolled on the day that they sign consent. Core biopsy specimens will be archived during screening. The screening visit can occur on the same day as the first injection (C1D1). The patient will be seen in clinic and complete all screening procedures and labs. Once the patient is deemed eligible for enrollment and has provided informed consent, the patient will be able to receive the first injection that same day. We will record the vital signs, ECOG performance status, adverse event review, and concomitant medication review from that day for both the screening visit and the C1D1 visit.
6.0 trial flow chart 6.1
7.1.6.2 treatment period

Patients will be seen in clinic on the day of each treatment administration. For the dose escalation cohort, there will two drug administrations, 3 weeks (+/- 1 week) apart. In the expansion cohort, for patients on the treatment arm, there will be four drug administrations, 3 weeks (+/- 1 week)
apart. In both cohorts, surgery will occur 3 weeks (+/- 2 weeks) after the 2nd or 4th dose of intralesional pembrolizumab
6.0 trial flow chart 6.1
7.1.6.3 post-treatment visits

Patients will be seen on a standard post-operative visit (2-3 weeks after surgery).
6.0 trial flow chart 6.1
7.1.5.3.1 safety follow-up visit

The mandatory Safety Follow-Up Visit should be conducted approximately 8 weeks post-surgery (+/- 2 weeks). All non-surgically-related AEs that occur prior to the Safety Follow-Up Visit should be recorded. Subjects with an AE of Grade > 1 will be followed until the resolution of the AE to Grade 0-1 or until the beginning of a new anti-neoplastic therapy, whichever occurs first. SAEs that occur within 90 days of the end of treatment or before initiation of a new anti-cancer treatment should also be followed and recorded.
6.0 trial flow chart 6.1
7.1.6.4 follow-up visits 7.1.6.4.1 survival

This is a short term study using surgical endpoints. We will follow patients for toxicity (8 weeks post-surgery (+/- 2 weeks)).
6.0 trial flow chart 6.1
7.2 assessing and recording adverse

An adverse event (also known as an adverse experience) is defined as any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug related. More specifically, an adverse event (can be any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the use of a drug, without any judgment about causality. An adverse event can arise from any use of the drug (e.g., off-label use, use in combination with another drug) and from any route of administration, formulation, or dose, including an overdose.
6.0 trial flow chart 6.1
7.2.1.2 adverse reaction

An adverse reaction is defined as any adverse event caused by the use of a drug. Adverse reactions are a subset of all suspected adverse reactions for which there is reason to conclude that the drug caused the event.
6.0 trial flow chart 6.1
7.2.1.3 suspected

A suspected adverse reaction is defined as any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, "reasonable possibility" indicates that there is evidence to suggest a causal relationship between  
the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than an adverse reaction.
6.0 trial flow chart 6.1
7.2.1.4 unexpected

An adverse event or suspected adverse reaction is considered *unexpected* if it is not listed in the investigator brochure or package insert(s), or is not listed at the specificity or severity that has been observed, or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application. "Unexpected," as used in this definition, also refers to adverse events or suspected adverse reactions that are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned as occurring with the particular drug under investigation. Adverse events that would be anticipated to occur as part of the disease process are considered unexpected for the purposes of reporting because they would not be listed in the investigator brochure. For example, a certain number of non-acute deaths in a cancer trial would be anticipated as an outcome of the underlying disease, but such deaths would generally not be listed as a suspected adverse reaction in the investigator brochure. Some adverse events are listed in the Investigator Brochure as occurring with the same class of drugs, or as anticipated from the pharmacological properties of the drug, even though they have not been observed with the drug under investigation. Such events would be considered *unexpected* until they have been observed with the drug under investigation. For example, although angioedema is anticipated to occur in some patients exposed to drugs in the ACE inhibitor class and angioedema would be described in the investigator brochure as a class effect, the first case of angioedema observed with the drug under investigation should be considered *unexpected* for reporting purposes.
6.0 trial flow chart 6.1
7.2.1.5 serious

An adverse event or suspected adverse reaction is considered *serious* if, in the view of either the investigator or sponsor, it results in any of the following outcomes:  
Death Life-threatening adverse event Inpatient hospitalization or prolongation of existing hospitalization A persistent or significant incapacity or substantial disruption of the ability to conduct normal life function Congenital anomaly/birth defect
Important medical events that may not result in death, are life-threatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent Page 42 of 102  
one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.
6.0 trial flow chart 6.1
7.2.1.6 life-threatening

An adverse event or suspected adverse reaction is considered *life-threatening* if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death.
6.0 trial flow chart 6.1
7.2.2 recording of an adverse

All Grade 3 and above adverse events will be entered into OnCore®, whether or not the event is believed to be associated with use of the study drug. Data about these events and their severity will be recorded using the NCI CTCAE v4.0. If there are specific data plans to this study, such as Grade 1 & 2 AEs are also being entered into OnCore, describe that process here. The Investigator will assign attribution of the possible association of the event with use of the investigational drug, and this information will be entered into OnCore® using the classification system listed below:  
| Relationship                                  | Attribution                                  | Description                                       |
|-----------------------------------------------|----------------------------------------------|---------------------------------------------------|
| Unrelated                                     | The AE is clearly NOT related to the         |                                                   |
| Unrelated to investigational                  | intervention                                 |                                                   |
| drug/intervention                             | Unlikely                                     | The AE is doubtfully related to the  intervention |
| Possible                                      | The AE may be related to the intervention    |                                                   |
| Probable                                      | The AE is likely related to the intervention |                                                   |
| Related to investigational  drug/intervention | Definite                                     | The AE is clearly related to the intervention     |  
Signs or symptoms reported as adverse events will be graded and recorded by the Investigator according to the CTCAE. When specific adverse events are not listed in the CTCAE they will be graded by the Investigator as none, mild, moderate or *severe* according to the following grades and definitions:  
Grade 0 No AE (or within normal limits) Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not indicated Grade 2 Moderate; minimal, local, or noninvasive intervention (e.g., packing, cautery)
indicated; limiting age-appropriate instrumental activities of daily living (ADL)
Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care ADL
Grade 4: Life-threatening consequences; urgent intervention indicated Grade 5: Death related to AE
6.0 trial flow chart 6.1
7.2.3 follow-up of adverse events

All adverse events will be followed with appropriate medical management until resolved. Patients removed from study for unacceptable adverse events will be followed until resolution or stabilization of the adverse event. For selected adverse events for which administration of the investigational drug was stopped, a re-challenge of the subject with the investigational drug may be conducted if considered both safe and ethical by the Investigator.
6.0 trial flow chart 6.1
7.2.4 adverse events monitoring

All adverse events, whether or not unexpected, and whether or not considered to be associated with the use of the study drug, will be entered into OnCore®, as noted above.  
The Investigator will assess all adverse events and determine reportability requirements to the UCSF Data and Safety Monitoring Committee (DSMC) and UCSF's Institutional Review Board, the Institutional Review Board (IRB); and, when the study is conducted under an Investigational New Drug Application (IND), to the Food and Drug Administration (FDA) if it meets the FDA reporting criteria. [The manufacturer and/or grant sponsor may also need to be notified, as applicable.]
All adverse events entered into OnCore® will be reviewed by the Helen Diller Family Comprehensive Cancer Center Site Committee on a weekly basis. The Site Committee will review and discuss at each weekly meeting the selected toxicity, the toxicity grade, and the attribution of relationship of the adverse event to the administration of the study drug(s). All grade(s) 3-5 adverse events entered into OnCore® will be reviewed on a monthly basis at the Site Committee meetings. The Site Committee will review and discuss the selected toxicity, the toxicity grade, and the attribution of relationship of the adverse event to the administration of the study drug(s). In addition, all suspected adverse reactions considered "serious" entered into OnCore®, will be reviewed and monitored by the Data and Safety Monitoring Committee on an ongoing basis and discussed at DSMC meetings, which take place every six weeks. For a detailed description of the Data and Safety Monitoring Plan for a Multicenter Phase 2 or 3 Institutional Study at the Helen Diller Comprehensive Cancer Center please refer Appendix 4 Multicenter Institutional Studies.
6.0 trial flow chart 6.1
7.2.5 expedited reporting reporting to

If a death occurs during the treatment phase of the study or within 30 days after the last administration of the study drug(s) and it is determined to be related either to the study drug(s) or to a study procedure, the Investigator or his/her designee must notify the DSMC Chair (or qualified alternate) within 1 business day of knowledge of the event. The contact may be by phone or email.
6.0 trial flow chart 6.1
reporting to ucsf institutional review

The Principal Investigator must report events meeting the UCSF IRB definition of "Unanticipated Problem" (UP) within 10 business days of his/her awareness of the event.
6.0 trial flow chart 6.1
expedited reporting to the food

If the study is being conducted under an IND, the Sponsor-Investigator is responsible for determining whether or not the suspected adverse reaction meets the criteria for expedited reporting in accordance with Federal Regulations (21 CFR §312.32). The Investigator must report in an IND safety report any suspected adverse reaction that is both serious and unexpected. The Sponsor-Investigator needs to ensure that the event meets all three definitions:  
Suspected adverse reaction - A suspected adverse reaction is defined as any adverse event for which there is a reasonable possibility that the treatment or procedure caused the adverse event. For the purposes of IND safety reporting, "reasonable possibility" indicates that there is evidence to suggest a causal relationship between the treatment or procedure and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than an adverse reaction.  
Unexpected - An adverse event or suspected adverse reaction is considered *unexpected* if it is not listed in the investigator brochure or package insert(s), or is not listed at the specificity or severity that has been observed, or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application.
"Unexpected," as used in this definition, also refers to adverse events or suspected adverse reactions that are mentioned in the investigator brochure as occurring with a class of treatment or procedure or as anticipated from the pharmacological properties of the treatment or procedure, but are not specifically mentioned as occurring with the particular treatment or procedure under investigation.  
Adverse events that would be anticipated to occur as part of the disease process are considered *unexpected* for the purposes of reporting because they would not be listed in the investigator brochure. For example, a certain number of non-acute deaths in a cancer trial would be anticipated as an outcome of the underlying disease, but such deaths would generally not be listed as a suspected adverse reaction in the investigator brochure. Some adverse events are listed in the Investigator's Brochure as occurring with the same class of treatment, or as anticipated from the pharmacological properties of the drug, even though they have not been observed with the drug under investigation. Such events would be considered *unexpected* until they have been observed with the drug under investigation. For example, although angioedema is anticipated to occur in some patients exposed to drugs in the ACE inhibitor class and angioedema would be described in the investigator brochure as a class effect, the first case of angioedema observed with the drug under investigation should be considered *unexpected* for reporting purposes.  
Serious - An adverse event or suspected adverse reaction is considered *serious* if, in the view of either the investigator or sponsor, it results in any of the following outcomes:
Death Life-threatening adverse event Inpatient hospitalization or prolongation of existing hospitalization A persistent or significant incapacity or substantial disruption of the ability to conduct normal life function Congenital anomaly/birth defect Important medical events that may not result in death, are life-threatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse. If the adverse event does not meet all three of the definitions, it should not be submitted as an expedited IND safety report.  
The timeline for submitting an IND safety report to FDA is no later than **15 calendar days** after the Investigator determines that the suspected adverse reaction qualifies for reporting (21 CFR 312.32(c)(1)). Any unexpected fatal or life-threatening suspected adverse reaction will be reported to FDA no later than **7 calendar days** after the Investigator's initial receipt of the information (21 CFR
312.32(c)(2)). Any relevant additional information that pertains to a previously submitted IND safety report will be submitted to FDA as a Follow-up IND Safety Report without delay, as soon as the information is available (21 CFR 312.32(d)(2)).
6.0 trial flow chart 6.1
expedited reporting to merck.

Investigators must report all SAEs to Merck within 24 hours of becoming aware of the event.
6.0 trial flow chart 6.1
7.2.5.1 pembrolizumab overdose

For purposes of this trial, an overdose of pembrolizumab will be defined as any dose of 1,000 mg or greater (≥5 times the indicated dose). No specific information is available on the treatment of overdose of pembrolizumab. Appropriate supportive treatment should be provided if clinically indicated. In the event of overdose, the subject should be observed closely for signs of toxicity. Appropriate supportive treatment should be provided if clinically indicated. If an adverse event(s) is associated with ("results from") the overdose of a Merck product, the adverse event(s) is reported as a serious adverse event, even if no other seriousness criteria are met. If a dose of Merck's product meeting the protocol definition of overdose is taken without any associated clinical symptoms or abnormal laboratory results, the overdose is reported as a nonserious Event of Clinical Interest (ECI), using the terminology "accidental or intentional overdose without adverse effect." All reports of overdose with and without an adverse event must be reported within 24 hours to the Sponsor and within 2 working days hours to Merck Global Safety.
6.0 trial flow chart 6.1
7.2.5.2 reporting of pregnancy and

Although pregnancy and infant exposure during breast feeding are not considered adverse events, it is the responsibility of investigators or their designees to report any pregnancy or lactation in a participant (spontaneously reported to them) that occurs during the study. Pregnancies and infant exposures during breastfeeding that occur after the consent form is signed but before treatment must be reported by the investigator if they cause the participant to be excluded from the trial, or are the result of a protocol-specified intervention, including but not limited to washout or discontinuation of usual therapy, diet, placebo treatment or a procedure. Pregnancies and infant exposures during breastfeeding that occur from the time of treatment through 120 days following cessation of Sponsor's product, or 30 days following cessation of treatment if the participant initiates new anticancer therapy, whichever is earlier, must be reported by the investigator. All reported pregnancies must be followed to the completion/termination of the pregnancy. Pregnancy outcomes of spontaneous abortion, missed abortion, benign hydatidiform mole, blighted ovum, fetal death, intrauterine death, miscarriage and stillbirth must be reported as serious events (Important Medical Events). If the pregnancy continues to term, the outcome (health of infant) must also be reported. Such events must be reported within 24 hours to the Sponsor and within 2 working days 7.2.5.3 **Immediate Reporting of Adverse Events to the Sponsor and to Merck**
6.0 trial flow chart 6.1
7.2.5.4 serious adverse events

A serious adverse event is any adverse event occurring at any dose or during any use of Merck's product that:
Results in death; Is life threatening; Results in persistent or significant disability/incapacity; Results in or prolongs an existing inpatient hospitalization; Is a congenital anomaly/birth defect; Is any other important medical event
**Note:** In addition to the above criteria, adverse events meeting either of the below criteria, although not serious per ICH definition, are reportable to the Merck in the same timeframe as SAEs to meet certain local requirements. Therefore, these events are considered serious by Merck for collection purposes.  
Is a new cancer (that is not a condition of the study); Is associated with an overdose;
Refer to Table 7 for additional details regarding each of the above criteria. Any serious adverse event, or follow up to a serious adverse event, including death due to any cause other than progression of the cancer under study that occurs to any subject from the time the consent is signed through 90 days following cessation of treatment, or the initiation of new anticancer therapy, whichever is earlier, whether or not related to Merck product, must be reported within 24 hours to the Sponsor and within 2 working days to Merck Global Safety.  
Non-serious Events of Clinical Interest will be forwarded to Merck Global Safety and will be handled in the same manner as SAEs. Additionally, any serious adverse event, considered by an investigator who is a qualified physician to be related to Merck product that is brought to the attention of the investigator at any time outside of the time period specified in the previous paragraph also must be reported immediately to the Sponsor and to Merck.
6.0 trial flow chart 6.1
sae reports and any other

A copy of all 15 Day Reports and Annual Progress Reports is submitted as required by FDA, European Union (EU), Pharmaceutical and Medical Devices agency (PMDA) or other local Page 48 of 102  
regulators. Investigators will cross reference this submission according to local regulations to the Merck Investigational Compound Number (IND, CSA, etc.) at the time of submission. Additionally investigators will submit a copy of these reports to Merck & Co., Inc.  
at the time of submission to FDA.  
All subjects with serious adverse events must be followed up for outcome.
6.0 trial flow chart 6.1
7.2.5.5 events of clinical interest

Selected non-serious and serious adverse events are also known as Events of Clinical Interest (ECI) and must be recorded as such on the Adverse Event case report forms/worksheets and reported within 24 hours to the Sponsor and within 2 working days to Merck Global Safety. For the time period beginning when the consent form is signed until treatment, any ECI, or follow up to an ECI, that occurs to any participant must be reported within 2 working days to Merck Global Safety if it causes the participant to be excluded from the trial, or is the result of a protocolspecified intervention, including but not limited to washout or discontinuation of usual therapy, diet, placebo treatment or a procedure. For the time period beginning at treatment through 90 days following cessation of treatment, or 30 days following cessation of treatment if the participant initiates new anticancer therapy, whichever is earlier, any ECI, or follow up to an ECI, whether or not related to Merck product, must be reported within 2 working days to Merck Global Safety. Events of clinical interest for this trial include: 1. an overdose of Merck product, as defined in Section 7.2.5.1 - Definition of an Overdose for This Protocol and Reporting of Overdose to the Sponsor, that is not associated with clinical symptoms or abnormal laboratory results. 2. an elevated AST or ALT lab value that is greater than or equal to 3X the upper limit of normal and an elevated total bilirubin lab value that is greater than or equal to 2X the upper limit of normal and, at the same time, an alkaline phosphatase lab value that is less than 2X the upper limit of normal, as determined by way of protocol-specified laboratory testing or unscheduled laboratory testing.*
*Note: These criteria are based upon available regulatory guidance documents. The purpose of the criteria is to specify a threshold of abnormal hepatic tests that may require an additional evaluation for an underlying etiology. The trial site guidance for assessment and follow up of these criteria can be found in the Investigator Trial File Binder (or equivalent).
6.0 trial flow chart 6.1
7.2.6 evaluating adverse events

An investigator who is a qualified physician will evaluate all adverse events according to the NCI Common Terminology for Adverse Events (CTCAE), version 4.0. Any adverse event which changes CTCAE grade over the course of a given episode will have each change of grade recorded on the adverse event case report forms/worksheets. All adverse events regardless of CTCAE grade must also be evaluated for seriousness.
6.0 trial flow chart 6.1
table 7. evaluating adverse events

| An investigator who is a qualified physician, will evaluate all adverse events as to: V4.0  CTCAE  Grading Grade 1 Mild; asymptomatic or mid symptoms; clinical or diagnostic observations only;  intervention not indicated. Grade 2 Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate  instrumental ADL. Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or  prolongation or hospitalization indicated; disabling; limiting self-care ADL. Grade 4 Life threatening consequences; urgent intervention indicated. Grade 5 Death related to AE Seriousness A serious adverse event is any adverse event occurring at any dose or during any use of Merck product that: †Results in death; or †Is life threatening; or places the subject, in the view of the investigator, at immediate risk of death from the event  as it occurred (Note: This does not include an adverse event that, had it occurred in a more severe form, might have  caused death.); or †Results in a persistent or significant disability/incapacity (substantial disruption of one's ability to conduct  normal life functions); or †Results in or prolongs an existing inpatient hospitalization (hospitalization is defined as an inpatient admission,  regardless of length of stay, even if the hospitalization is a precautionary measure for continued observation. (Note:  Hospitalization [including hospitalization for an elective procedure] for a preexisting condition which has not  worsened does not constitute a serious adverse event.); or †Is a congenital anomaly/birth defect (in offspring of subject taking the product regardless of time to diagnosis);or Is a new cancer; (that is not a condition of the study) or Is an overdose (whether accidental or intentional). Any adverse event associated with an overdose is considered a  serious adverse event. An overdose that is not associated with an adverse event is considered a non-serious event of  clinical interest and must be reported within 24 hours. Other important medical events that may not result in death, not be life threatening, or not require hospitalization  may be considered a serious adverse event when, based upon appropriate medical judgment, the event may  jeopardize the subject and may require medical or surgical intervention to prevent one of the outcomes listed  previously (designated above by a †). Duration Record the start and stop dates of the adverse event. If less than 1 day, indicate the appropriate length of time and  units Action taken Did the adverse event cause the Merck product to be discontinued? Relationship  Did the Merck product cause the adverse event? The determination of the likelihood that the Merck product caused  to test drug  the adverse event will be provided by an investigator who is a qualified physician. The investigator's signed/dated  initials on the source document or worksheet that supports the causality noted on the AE form, ensures that a  medically qualified assessment of causality was done. This initialed document must be retained for the required  regulatory time frame. The criteria below are intended as reference guidelines to assist the investigator in assessing  the likelihood of a relationship between the test drug and the adverse event based upon the available information.  The following components are to be used to assess the relationship between the Merck product and the AE;  the greater the correlation with the components and their respective elements (in number and/or intensity), the more  likely the Merck product caused the adverse event (AE): Exposure Is there evidence that the subject was actually exposed to the Merck product such as: reliable  history, acceptable compliance assessment (pill count, diary, etc.), expected pharmacologic  effect, or measurement of drug/metabolite in bodily specimen? Time Course Did the AE follow in a reasonable temporal sequence from administration of the Merck  product?  Is the time of onset of the AE compatible with a drug-induced effect (applies to trials with  investigational medicinal product)? Likely Cause Is the AE not reasonably explained by another etiology such as underlying disease, other  drug(s)/vaccine(s), or other host or environmental factors   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Relationship                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | The following components are to be used to assess the relationship between the test drug and the AE:  (continued)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| to Merck  product (continued)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Dechallenge                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Was the Merck product discontinued or dose/exposure/frequency reduced? If yes, did the AE resolve or improve? If yes, this is a positive dechallenge. If no, this is a negative dechallenge. (Note: This criterion is not applicable if: (1) the AE resulted in death or permanent disability;  (2) the AE resolved/improved despite continuation of the Merck product; or (3) the trial is a  single-dose drug trial); or (4) Merck product(s) is/are only used one time.) |
| Rechallenge                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Was the subject re-exposed to the Merck product in this study? If yes, did the AE recur or worsen? If yes, this is a positive rechallenge. If no, this is a negative rechallenge. (Note: This criterion is not applicable if: (1) the initial AE resulted in death or permanent  disability, or (2) the trial is a single-dose drug trial); or (3) Merck product(s) is/are used only  one time). NOTE: IF A RECHALLENGE IS PLANNED FOR AN ADVERSE EVENT WHICH WAS  SERIOUS AND WHICH MAY HAVE BEEN CAUSED BY THE MERCK PRODUCT, OR  IF REEXPOSURE TO THE MERCK PRODUCT POSES ADDITIONAL POTENTIAL  SIGNIFICANT RISK TO THE SUBJECT, THEN THE RECHALLENGE MUST BE  APPROVED IN ADVANCE BY THE U.S. CLINICAL MONITOR AS PER DOSE  MODIFICATION GUIDELINES IN THE PROTOCOL. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| Consistency  with Trial  Treatment  Profile                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Is the clinical/pathological presentation of the AE consistent with previous knowledge  regarding the Merck product or drug class pharmacology or toxicology?                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
| The assessment of relationship will be reported on the case report forms /worksheets by an investigator who is a qualified physician  according to his/her best clinical judgment, including consideration of the above elements. Record one of the following Use the following scale of criteria as guidance (not all criteria must be present to be  indicative of a Merck product relationship). Yes, there is a reasonable  possibility of Merck product  relationship. There is evidence of exposure to the Merck product. The temporal sequence of the AE onset  relative to the administration of the Merck product is reasonable. The AE is more likely  explained by the Merck product than by another cause. No, there is not a reasonable  possibility Merck product  relationship Subject did not receive the Merck product OR temporal sequence of the AE onset relative to  administration of the Merck product is not reasonable OR there is another obvious cause of  the AE. (Also entered for a subject with overdose without an associated AE.) |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
6.0 trial flow chart 6.1
7.2.7 sponsor responsibility for reporting

All Adverse Events will be reported to regulatory authorities, IRB/IECs and investigators in accordance with all applicable global laws and regulations.
6.0 trial flow chart 6.1
8.0 statistical analysis plan 8.1

The primary endpoint of this study is the MTD of two intralesional injections of pembrolizumab in patients with DCIS. The primary safety endpoint is DLT. The MTD is defined as the dose at which no more than 1/6 patients experience a DLT. Safety and tolerability will also be assessed by clinical review of all relevant parameters including adverse events (AEs), laboratory tests, and physical exam.  
Adverse events will be summarized as counts and frequencies for each dose level. Other safety endpoints will be summarized as appropriate. These data are descriptive in nature and statistical analysis tools will not be required. The recommended expansion dose will be explored in an additional cohort of 30 patients with DCIS (20 to receive pembrolizumab and 10 untreated control subjects).