Exploring the Utility of a Novel BRAF Test in Patients With Melanoma
Conditions: Melanoma
Sponsor: Massachusetts General Hospital

 
 
 
 
STATUS PAGE 
PROTOCOL 12-488 
 
 
 
 
Closed To New Accrual 
 
 
Closure Effective Date: 03/29/2017 
Reason: Study Accrual Goal Met 
 
 
No new subjects may be enrolled in the study- as described above. 
Any questions regarding this closure should be directed to the study’s 
Principal Investigator 

DFCI Protocol No: 12-488
Front Sheet
05/09/2018 02:06 PM
Report Generated:
Exploring the Utility of a Novel BRAF Test in Patients with Melanoma
Title:
Overall Principal Investigator
Email
Institution
Sullivan, Ryan Joseph
rsullivan7@partners.org
Massachusetts General Hospital
Overall Institution:
Massachusetts General Hospital
Primary Contact(s) for Questions
Email
Phone
Lawless, Aleigha
alawless@partners.org
Site Responsible PI(s)
Email
Institution
Hodi, Frank Stephen
FHODI@PARTNERS.ORG
Dana-Farber Cancer Institute
McDermott, David F
dmcdermo@bidmc.harvard.edu
Beth Israel Deaconess Medical Center
Total Study-Wide Enrollment Goal:
220
Total DF/HCC Estimated Enrollment Goal:
220
Phase: Not Applicable
Age:
Adults
Age Ranges:
18+
Will all subjects be recruited from pediatric clinics?
CTEP Study:
No
Management Group(s):
Primary Management Group:
DF/HCC Melanoma
BIDMC Biologics
(Kidney/Melanoma/Cutaneous)
DF/HCC Melanoma
DFCI/BWH Cutaneous Oncology
MGH Melanoma
MGH Regulatory Coordinators
Sponsor Name
Sponsor Protocol No
Roles
Grant Number(s)
DF/HCC Investigator
Regulatory
Dana-Farber/Harvard Cancer Center
Funding
ASCO Foundation
Funding
Investigational Drug?
This study uses a Drug/Biologic, but an IND was not submitted.
Drug(s), Biologic(s):
Investigational Device?
This study uses an Investigational Device, but an IDE was not submitted.
Device Name:
Risk Category:
Minimal Risk
Human Material Banking; Human Material Collection; Medical Record Review
Protocol Involves:
Date Range: (Medical Record Review and Specimen Collection studies)
IRB of Record:
Participating Sites under the DFCI IRB
Institution:
Beth Israel Deaconess Medical Center
Dana-Farber Cancer Institute
Massachusetts General Hospital

05/09/2018 02:06 PM
Report Generated:
Participating Institutions Under Other IRB
None

Log Sheet 
Page 1 of 1 
 
Protocol Number: 12-488 
 
Approval Date:    03/11/13   (IRB meeting date when protocol/consent  
 approved or conditionally approved) 
 
Activation Date:    05/15/13   (Date when protocol open to patient entry) 
 
Approval signatures are on file in the Office for Human Research Studies, tel. 617-632-3029. 
 
Date 
Posted 
Revised Sections 
IRB Approval 
Date 
OHRS 
Version Date 
06/19/13 
Front Sheet replaced due to Amendment #1 
06/18/13 
N/A 
09/30/13 
Delayed Activation Alert Page removed: DFCI now 
ready for activation (note: previously activated at MGH 
& BWH on  05/15/13) 
N/A 
N/A 
02/11/14 
Study renewal/ Consent Form footer replaced due to 
Continuing Review #1 
02/10/14 
N/A 
11/06/14 
Consent Form and Front Sheet replaced due to 
Amendment #2 
10/30/14 
10/31/14 
02/11/15 
ON HOLD:  All research must stop due to lapsed 
Continuing Review.  Study expired 02/10/15 
N/A 
N/A 
02/11/15 
Remove Hold. Study renewal/ Consent Form footer 
replaced due to Continuing Review #2 
01/14/15 
N/A 
Date 
Posted 
Revised Sections 
IRB Approval 
Date 
OnCore 
Version Date 
01/13/16 
Study renewal/ Consent Form footer replaced due to 
Continuing Review #3 
01/10/16 
01/13/16 
04/27/16 
Protocol, Eligibility Checklist, Consent Form and Front 
Sheet replaced due to Amendment #3 
04/07/16 
04/27/16 
Date 
Posted 
Revised Sections 
Approved 
Date  
Version Date 
(OnCore) 
01/05/17 
Study renewal/ Consent Form footer replaced due to 
Continuing Review #4 
12/21/2016 
 
12/21/2016 
03/30/17 
Study closure – accrual goal met 
03/29/17 
N/A 
12/13/17 
Study renewal/Consent Form footer replaced due to 
Continuing Review #5 
12/11/17 
12/11/17 
03/05/2018 
No change to online documents per Amendment #4 
03/05/2018 
n/a 
05/10/2018 
Front Sheet, Protocol replaced per Amendment #5 
04/30/2018 
N/A 
11/23/2018 
Study renewal/Consent Form footer replaced per 
Continuing Review #6 
11/21/2018 
11/21/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Page 1 
 
 
DF/HCC Protocol #: 12-488 
 
Title:  Exploring the Utility of a Novel BRAF Test in Patients with Melanoma 
 
Coordinating Center: 
 
 
Massachusetts General Hospital 
 
Principal Investigator (PI): 
Ryan J Sullivan, MD 
 
Massachusetts General Hospital 
 
RSULLIVAN7@PARTNERS.ORG 
 
Other Investigators: 
Dora Dias-Santagota, PhD, Massachusetts General Hospital 
 
Lyn Duncan, MD, Massachusetts General Hospital 
 
Donald Lawrence, M.D., Massachusetts General Hospital 
 
Keith Flaherty, M.D., Massachusetts General Hospital 
 
Krista Rubin, N.P., Massachusetts General Hospital 
 
Riley Fadden, N.P., Massachusetts General Hospital 
 
Alex Haynes, MD, Massachusetts General Hospital 
 
Genevieve Boland, MD, PhD, Massachusetts General Hospital 
 
James Cusack, MD, Massachusetts General Hospital 
 
Kenneth K Tanabe, MD, Massachusetts General Hospital 
Hensin Tsao, MD, Massachusetts General Hospital 
 
F. Stephen Hodi, MD, Dana Farber Cancer Institute 
 
Rizwan Haq, MD, Dana Farber Cancer Institute 
 
Patrick Ott, MD, Dana Farber Cancer Institute 
 
Elizabeth Buchbinder, MD, Dana Farber Cancer Institute 
 
Hilary Donahue, PA-C, Dana Farber Cancer Institute 
 
David McDermott, MD, Beth Israel Deaconess Medical Center 
 
James W. Mier, MD, Beth Israel Deaconess Medical Center 
 
Statistician: 
 
Study Coordinator:  
Anita Giobbie-Hurder 
Aleigha Lawless 
Dana Farber Cancer Institute                                Massachusetts General Hospital 
 
 
 
 
 
      alawless@PARTNERS.ORG 
 
Agent(s): No therapeutics to be administered under this protocol 
 
Protocol Type / Version # / Version Date: 
Version 4 / April 17, 2018 
 

DF/HCC Protocol #: 12-488 
Protocol Version Date: 17Apr2018 
 
2 
 
SCHEMA 
 
Primary Objective 
 
         Advanced Melanoma                                       Stage II/resected Stage III 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
No BRAF 
mutation 
via tissue 
analysis 
No further 
follow-up 
BRAF mutation via 
tissue analysis 
BRAF 
mutation 
No BRAF 
mutation 
Concordance of 
tissue and blood 
BRAF status 
Standard BRAF 
tissue analysis 
BRAF blood 
assay 
BRAF blood 
assay 
Pharmacodynamics 
study 
No BRAF 
mutation 
via tissue 
analysis 
No further 
follow-up 
BRAF mutation via 
tissue analysis 
BRAF 
mutation 
No BRAF 
mutation 
Concordance of 
tissue and blood 
BRAF status 
Standard BRAF 
tissue analysis 
BRAF blood 
assay 
BRAF blood 
assay 
Prognostic and 
predictive studies 

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3 
TABLE OF CONTENTS 
SCHEMA .........................................................................................................................................2 
1. 
OBJECTIVES ......................................................................................................................5 
1.1 
Study Design ............................................................................................................5 
1.2 
Primary Objectives...................................................................................................5 
1.3 
Secondary Objectives...............................................................................................5 
2. 
BACKGROUND .................................................................................................................6 
2.1 
Study Disease ...........................................................................................................6 
2.2 
IND Agent ................................................................................................................7 
2.3 
Rationale ..................................................................................................................7 
3. 
PARTICIPANT SELECTION ...........................................................................................10 
3.1 
Eligibility Criteria ..................................................................................................10 
3.2 
Exclusion Criteria ..................................................................................................11 
3.3 
Inclusion of Women and Minorities ......................................................................11 
4. 
REGISTRATION PROCEDURES ...................................................................................11 
4.1 
General Guidelines for DF/HCC Institutions ........................................................12 
4.2 
Registration Process for DF/HCC Institutions .......................................................12 
4.3 
General Guidelines for Other Investigative Sites ..................................................12 
4.4 
Registration Process for Other Investigative Sites.................................................12 
5. 
STUDY DESIGN...............................................................................................................12 
5.1 
Sample Collection ..................................................................................................12 
5.2 
Data Collection ......................................................................................................13 
5.3 
Criteria for Taking a Participant Off Study ...........................................................14 
5.4 
Duration of Follow Up ...........................................................................................14 
6. 
ADVERSE EVENTS .........................................................................................................14 
6.1 
Expected Toxicities ................................................................................................14 
6.2 
Adverse Event Reporting .......................................................................................14 
7. 
MEASUREMENT OF EFFECT........................................................................................15 
8. 
DATA REPORTING / REGULATORY REQUIREMENTS ...........................................15 
8.1 
Data Reporting .......................................................................................................15 
8.2 
Data Safety Monitoring..........................................................................................15 
8.3 
Protocol Review and Amendments ........................................................................15 
8.4 
Ethics and Good Clinical Practice (GCP) ..............................................................16 
8.5 
Study Documentation.............................................................................................16 
8.6 
Records Retention ..................................................................................................16 
9. 
STATISTICAL CONSIDERATIONS...............................................................................17 
9.1 
Study Design/Endpoints.........................................................................................17 

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10. 
PUBLICATION PLAN .....................................................................................................21 
REFERENCES ..............................................................................................................................22 

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1. OBJECTIVES 
 
Primary  
 
To determine the specificity and sensitivity of the blood based assay vis-à-vis tissue 
based BRAF analysis in patients with advanced and high-risk melanoma. 
Secondary  
1. To explore the pharmacodynamic effects of MAPK pathway inhibitors (including 
selective BRAF inhibitors, MEK inhibitors, and ERK inhibitors) utilizing pre- and 
on-treatment peripheral blood BRAFV600E mutational testing in patients with 
advanced melanoma (non-resectable Stage III or Stage IV). 
2. To define the prognostic value of peripheral blood BRAFV600 mutational testing in 
patients with Stage II and resectable Stage III melanoma. 
 
1.1 
Study Design 
 
For the purposes of serum and plasma isolation optimization, patients who have agreed to 
participate in the study will donate no more than 20cc of peripheral blood (hereafter referred to 
as 1 sample) per timepoint. 10-20 ml blood will be acquired via standard venipuncture for serum 
and peripheral blood lymphocyte extraction at baseline and at varying intervals of time for each 
patient cohort. Amount of blood collected will follow the DF/HCC Guidance for Maximum 
Blood Draw for Research Purposes. 
 
Any patient may refuse a research blood draw at any time while on study, or may refuse to 
continue donating samples at any time and may withdraw consent from participation. Subjects 
will receive no compensation for participation in this study. All unused collected blood will be 
saved for possible use in future related analyses. 
 
1.2 
Primary Objectives 
 
To determine the sensitivity and specificity of peripheral blood BRAFV600 mutation detection in 
patients with melanoma, we will analyze samples from two distinct cohorts of patients: 1) 
Patients with advanced melanoma; and 2) Patients with resected, Stage II or Stage III disease.  A 
total of 110 patients in each cohort will be recruited to participate.  Peripheral blood lymphocytes 
(PBL) and serum will be isolated from blood. RNA will be isolated from tissue and PBL/serum 
and BRAFV600 analysis will be performed. Patients will be identified as being either positive or 
negative for the BRAFV600 mutation based on the results of the tissue testing. The tumor tissue 
(either primary tumor or nodal/systemic metastasis) from each patient with advanced disease, or 
from all patients with Stage II or resectable Stage III disease with adequate tissue, will be 
analyzed in a CLIA-approved laboratory for BRAFV600 sequencing analysis, either at the MGH 
TRL or at Clarient Laboratories using the FDA-approved Cobas® test. The results of the tissue-
based and peripheral blood test will be compared and percent concordance will be determined.  
 
1.3 
Secondary Objectives 
 

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6 
1. To explore the pharmacodynamic effects of MAPK-directed therapy on peripheral blood 
BRAFV600 mutation detection, we will test isolated PBL and serum from samples 
obtained from patients in the cohort with advanced melanoma prior to receiving treatment 
with MAPK-directed therapy or immunotherapy (either commercially or on protocol). 
Patients will have been previously identified as being positive for a BRAFV600 mutation 
based on the results of the testing of their tumor tissue. PBL and serum will be isolated 
from blood. RNA will then be isolated from tissue and PBL/serum and BRAFV600 
analysis will be performed as described above. Peripheral blood samples obtained prior to 
each cycle of MAPK-directed therapy or immunotherapy (either every three weeks or 
every four weeks depending on treatment protocol) will also be tested for BRAFV600 
RNA. Input RNA normalization will be carried out by using primers to a ribosomal 
protein, S-9, following the reverse transcription. Pre- and on-treatment BRAFV600 levels 
will be compared, and a blood-based response will be defined as a 50% or more decrease 
in signal based on acrylamide gel analysis. A blood-based progression will be defined as 
a doubling in signal based compared with nadir level on acrylamide gel analysis. A 
comparison of blood-based response or progression ( >50% improvement vs. <50% 
improvement; or 100% increase vs. < 100% increase) with clinical response (CR/PR by 
RECIST) and clinical progression (PD) will then be performed to assess concordance 
between changes over time with our assay and with imaging. 
 
2. To explore the prognostic and predictive ability of peripheral blood BRAFV600 mutation 
detection in the cohort of patients with Stage II or resected Stage III disease, we will test 
isolated PBL and serum from samples obtained from patients following complete 
resection of their disease but prior to receiving adjuvant therapy or embarking on 
expectant monitoring. To control for potential variations in circulating BRAF levels at 
various post-operative time points, patients must have their blood draw at least 4 weeks 
but no more than 8 weeks following their last surgery. Additionally, all patients with a 
thick primary melanoma (defined by AJCC as Breslow depth greater than 4 mm) or a 
clinically apparent/macroscopic involved lymph node, satellite lesion, or in-transit lesion 
will have tissue-based BRAF analysis performed either at the MGH TRL, at Clarient 
Laboratories using the FDA-approved Cobas® test, or in MGH pathology using 
BRAFV600 stain. PBL and serum will be isolated from blood collected in two 10 mL 
green top tubes on each patient and will then be placed in Trizol Reagent prior to RNA 
isolation. RNA will then be isolated from tissue and PBL/serum and BRAFV600 analysis 
will be performed as described above. 
 
 
2. BACKGROUND 
 
2.1 
Study Disease 
 
Metastatic melanoma is currently the 5th and 6th most common cancer in American men and 
women, respectively, and remains one of the few cancers with a rising incidence.(1) Over 9000 
people are expected to die in the United States in 2012 from this dreaded disease. Recent 
treatment advances have been made which has led to the FDA approval of both vemurafenib, a 
BRAF inhibitor, and ipilimumab, an immunotherapy, for the treatment of patients with advanced 

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7 
melanoma.(2-4) Unfortunately, the overwhelming majority of these patients still will die of their 
disease in spite of receiving these therapies.  
 
2.2 
IND Agent 
 
No therapeutics to be administered under this protocol  
 
2.3 
Rationale 
 
Strategies are underway to improve the effectiveness of BRAF-directed therapy and immune-
based therapy, and similar efforts are needed to develop biomarkers with which we may better 
select treatment for melanoma patients and follow patients while they are being treated. With 
BRAF-targeted therapy now established as a standard therapy for metastatic melanoma, the 
development of highly-sensitive, blood-based assays have the potential to greatly improve the 
care of patients with BRAF-mutant melanoma in four major ways. 
First, less than half of all patients with cutaneous melanoma have a tumor that harbors an 
oncogenic BRAF mutation.(5) In practice, current BRAF mutational analytic techniques are only 
capable of testing archival tissue and have limited sensitivity. Additionally, test results typically 
take two to three weeks, when factoring tissue acquisition and shipping issues, which delays the 
initiation of therapy for typically ill patients. Lastly, very often a patient who previously was 
diagnosed with an early stage melanoma who recurs will have a fine needle aspiration performed 
to confirm the diagnosis, and performing BRAF mutational analysis can be challenging due to 
the small amount of tissue that is obtained during such procedures. We believe that analyzing 
blood for the BRAF mutation will prove to be a more efficient method of determining a patient’s 
BRAF status and possibly a more reliable one. 
Second, tumor resistance to BRAF-directed therapy typically develops within six to eight months 
following initial tumor regression, but with a range of 2 months to 2 years.(2, 6-9) Importantly, 
each described mechanism involves the retention of the initiating BRAF mutation.(10-20) As the 
mechanisms of resistance are just now being elucidated, we feel that diagnostic assays which 
may identify developing resistance at an earlier time-point than standard clinical or radiographic 
assessments will have the ability to assist clinicians and researchers in determining the best next 
treatment option for patients whose disease is progressing on BRAF–directed therapy. This is 
particularly important due to the fact that a number of patients treated with BRAF inhibitors 
progress quite quickly following initial disease regression.(7) In these patients, it is typically 
difficult to transition to other therapies, such as ipilimumab, due to the rapid pace of growth in 
these resistant tumors.(21) While there is no current data which specifically supports such a 
strategy, it is thought that more advanced notice of disease progression when disease growth is 
more modest would allow for a more timely shift in treatment and improved benefit of next line 
therapy. Essentially, identifying a blood-based assay that can detect more subtle changes in 
disease burden than standard cross-sectional imaging would be requisite towards implementing 
this type of an approach. In addition, earlier detection of disease progression would also 
predictably allow investigators to analyze tumors or circulating tumor cells in these patients with 
a goal of determining the specific mechanism of resistance for an individual patient.  This would 
have the added value of predicting which next therapy would be most appropriate for any 

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8 
individual patient. 
Third, over the next 12 months, it is expected that multiple trials will open to test the 
effectiveness of BRAF-directed therapy in patients with resected, high-risk disease. In patients 
with resected disease, the current standard of care for adjuvant therapy is interferon alpha, which 
has reproducibly been shown to improve disease-free survival but not overall survival.(22) 
Further, while the development of serologic or clinical evidence of autoimmunity has been 
associated with a marked improvement in overall survival in patients treated with adjuvant 
interferon, there has never been a reliable pre-treatment biomarker that predicts which patients 
are more likely to benefit from this therapy.(23) It is critical that trials of adjuvant BRAF-
directed therapy test potential predictive biomarkers of benefit. One such biomarker would 
surely be a detectable BRAF mutation in tissue, as it is clear that patients with advanced 
melanoma only benefit from BRAF-directed therapy if there they have a tumor with a detectable 
BRAF mutation, however assessment of BRAF mutations in primary melanoma samples may be 
complicated by substantial tumor heterogeneity.(24) This may lead to both more false negatives 
and false positives, as it may not be clear which of the heterogeneous clones will ultimately 
establish metastasis. While it is uncertain whether detectable or high levels of circulating BRAF 
will be prognostic of a poor outcome or predictive of benefit to adjuvant BRAF-directed therapy, 
it is critical to evaluate both the predictive and prognostic qualities of blood-based BRAF assays 
in patients enrolled in adjuvant trials of BRAF inhibitors, as it is more likely that circulating cells 
of the subpopulations of primary melanomas are more reflective of the cells that will recur.  
Fourth, the recommended optimal follow up of patients who are currently rendered disease free 
with surgery for their melanoma is unknown.  Namely, the current NCCN guidelines do not 
recommend any blood analysis for such patients and only concede that considerations be made 
towards routine imaging in patients with higher risk disease (stage IIB-resected stage IV).(25) 
The development and validation of a blood-based prognostic biomarker would offer the potential 
to improve these guidelines and potential help direct radiographic imaging.  As an example, it is 
conceivable that the positive predictive value of cross-sectional imaging would be much higher 
in the context of a rising biomarker, much the way it is in patients with concerning symptoms, 
then when imaging is performed at randomly selected intervals.   
In summary, a blood-based BRAF mutational analysis has the potential to: 
1. Enhance and potentially replace tissue based BRAF mutation detection. 
2. Detect disease progression at an earlier time-point than imaging which may improve the 
effectiveness of next line therapy (given the lower tumor burden), and mechanism of 
resistance analysis for patients with BRAF mutant disease progressing on BRAF-directed 
therapy. 
3. Predict which patients respond to adjuvant, BRAF-directed therapy. 
4. Serve as a prognostic biomarker for patients with resected melanoma that may be followed 
serially and help clinicians choose which patients to perform diagnostic imaging. 
Mutational testing for BRAFV600 in tumor tissue has been well described using techniques such 
as bidirectional direct fluorescent sequencing and allele-specific polymerase chain reaction. The 
sensitivity of these assays, however is limited in that the assays in current use are only able to 

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9 
detect the mutation if the tumor cells constitute >5-10% of the specimen submitted for genetic 
analysis.(26) While this degree of sensitivity is typically sufficient to detect the presence of the 
BRAF mutation in a homogenous tumor nodule, this is likely not sensitive enough to detect a 
few tumor cells in the background of a high percentage of stromal or lymphatic elements, 
infiltrating lymphocytes, or peripheral blood cells.   
Recently, we have developed an RT-PCR-based technique that takes advantage of a unique 
restriction enzyme site present in wild-type (WT) but not the mutant BRAF to enrich the sample 
for the mutant form initially and then inserts a restriction site into the mutant but not WT BRAF 
to allow for detection and quantification of mutant BRAF.(27) As outlined in our original 
description of this technique and Figure 1, the protocol involves a series of PCR amplifications 
and restriction digestions that discriminate between WT and mutant BRAF at amino acid 
position 600. An initial RT-PCR is followed by digestion with TspR1 that preferentially digests 
the WT product but not the BRAFV600 PCR product. A nested PCR using the digested material 
follows. This PCR product is subjected to sequencing using a nested oligonucleotide. The PCR 
product is then subjected to a third PCR (product 140 bp) using a unique nested forward 
oligonucleotide that creates aXba 1 restriction site in the amplified product only within the 
mutant sequence. After an Xba 1 digest, the products (108 and 32 bp) are separated on 15% 
acrylamide gels.  After ethidium bromide staining the band at 108 bp is quantitated by 
densitometry. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In tissue, we utilized this assay to detect mutation at a two log-fold greater sensitivity (1:999) 
than standard assay and, with a slight modification of the protocol, are now able to increase the 
sensitivity to one-mutant cells in a million non-mutant cells (1:400,000). This level of detection 
is theoretically high enough to detect the mutation in circulating tumor cells (CTCs) which are 
reportedly found in patients with melanoma at a range of from 1-100 per million peripheral 
blood lymphocytes.(28) As a proof of principle, we initially detected the mutation in both the 
blood of patients with known tissue BRAFV600 mutation prior to their enrollment on the Phase 2 

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trial of vemurafenib and then in the blood of patients with resected stage III melanoma which 
was collected as part of the Harvard Skin SPORE blood and tissue banking protocol prior to the 
commencement of adjuvant therapy. 
The next step in the development of this assay is optimizing the quantification process and then 
validating its use in relevant patient cohorts.  One of the limitations of the current assay is that 
the insertion of the Xba1 one site during the tertiary PCR step only allows for assaying the 
BRAFV600E mutation specifically. While this is the most common oncogenic BRAFV600 mutation, 
other mutations such as V600D and V600K make up 2-20% of BRAFV600 mutations.(2, 5, 7, 29, 
30) As a result, we are currently looking into other methods of analyzing the data.  Specifically, a 
second-generation assay will maintain the initial TspR1 digestion step, as this is critical to 
maintaining the sensitivity of the assay.  As has been mentioned, direct sequencing is possible 
following the second PCR step.  While this step is unnecessary for detection and quantification, 
by applying next generation sequencing approaches, we believe that this will allow for the 
detection of non-V600E BRAF mutations while at the same time, possibly improve 
quantification of BRAF mutation levels. 
 
We propose to develop and standardize a test which can reliably detect the BRAFV600E mutation 
in patients with known tissue BRAFV600E mutation in the blood of patients with melanoma. We 
aim to correlate testing with worse clinical outcome when disease is resected and to correlate 
blood testing with disease response to MAPK pathway inhibitors in the advanced, as well as 
potentially the adjuvant, setting.  
 
 
3. PARTICIPANT SELECTION 
 
Participants must meet the following criteria on screening examination to be eligible to 
participate in the study: 
 
3.1 
Eligibility Criteria  
 
3.1.1 All participants must have biopsy-proven advanced (unresectable stage IIIC or Stage IV) 
or high-risk (Stage II or Stage III) malignant melanoma 
 
3.1.2 Participants must be 18 years of age or older 
 
3.1.3 Participant must have the ability to understand and willingness to sign a written informed 
consent document   
 
 
 
 
 
 
 

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3.2 
Exclusion Criteria 
 
3.2.1 Participants with a history of different malignancy are ineligible except for the following 
circumstances.  
• Non-metastatic prostate cancer, cervical cancer in situ, and basal cell or squamous 
cell carcinoma of the skin. 
• Individuals with a history of other malignancies are eligible if they have been 
disease-free for at least 2 years and are deemed by the investigator to be at low 
risk for recurrence of that malignancy.  
 
3.2.2 Participants with a known history of a different BRAF-mutant malignancy  
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial 
 
 
4. REGISTRATION PROCEDURES 
 
Participation is strictly voluntary. Patients coming to the participating sites that meet the 
eligibility criteria will be identified by physicians, study coordinator, research nurse, or 
other study team member. Potential participants will receive a description of the study, 
including the nature of participation, and the phone numbers for study contacts. Participants 
may be enrolled prospectively or retrospectively.   
 
Written informed consent will be obtained by a physician or other licensed study team 
member prior to enrollment into the study. All participants (except those who have previously 
signed consent to enroll in either the 02-017 or 11-181 protocols) must be provided a consent 
form describing this study and providing sufficient information for participants to make an 
informed decision about their participation in this study. Study coordinators will be allowed to 
obtain consent, as outlined in the November 2014 Guide to Human Research Activities, after 
receiving training from the principal investigator or by a sub-investigator following 
approval by the principal investigator.  
 
The formal consent of a participant, using the IRB approved consent form, must be 
obtained before the participant is involved in any study-related procedure. The consent 
form must be signed and dated by the participant or the participant’s legally authorized 
representative, and by the person obtaining the consent. The participant must be given a 
copy of the signed and dated consent document. The original signed copy of the consent 
document must be retained in the medical record or research file. 
 
 
This study has minimal to no risk to the study participants.   
 

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4.1 
General Guidelines for DF/HCC Institutions 
 
Institutions will register eligible participants in the Clinical Trials Management System (CTMS) 
OnCore. Registration must occur within 24 hours of the first blood sample being drawn. Any 
participant not consented to the protocol before protocol therapy begins will be considered 
ineligible and registration will be denied. 
 
An investigator will confirm eligibility criteria and a member of the study team will complete the 
protocol-specific eligibility checklist. 
 
Following registration, participants may continue protocol interventions. If a participant does not 
receive protocol interventions following registration, the participant’s registration on the study 
must be canceled. Registration cancellations must be made in OnCore as soon as possible.  
 
4.2 
Registration Process for DF/HCC Institutions 
 
DF/HCC Standard Operating Procedure for Human Subject Research Titled Subject Protocol 
Registration (SOP #: REGIST-101) must be followed.   
 
4.3 
General Guidelines for Other Investigative Sites 
 
Not Applicable. 
 
4.4 
Registration Process for Other Investigative Sites 
 
Not Applicable.  
 
 
5. STUDY DESIGN 
 
5.1 
Sample Collection 
 
Advanced Disease Cohort:  
 
Blood samples will be obtained on participants following signed informed consent.  For patients 
in the advanced melanoma cohort, a one-time blood sample will be obtained prior to the 
commencement of systemic therapy at a time when standard-of-care blood will be drawn.  While 
it is ideal to obtain samples prior to any systemic therapy, participants who have previously 
received therapy, have progressed, and are in need of additional therapy will be considered 
suitable for participation.  Participants with known BRAF mutation (determined by standard of 
care tissue testing) will undergo blood draws every 4 weeks (+/- 1 week) and at the time of 
disease progression.  
BRAF WT (tissue): One- time, pre-treatment blood draw 
BRAF Mut (tissue): Pre-treatment blood draw then every 4 weeks (+/- 1 week).  A 

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blood draw at time of progression will also be obtained. 
Stage II/ III Disease Cohort:  
Blood samples will be obtained on participants following signed informed consent. For patients 
in the high-risk stage II/III cohort, a blood draw will be performed 4-8 weeks after the 
completion of surgical management at the time of standard of care blood work. In participants 
who sign informed consent prior to definite surgical management, a pre-operative blood draw 
may be obtained at the time that standard of care pre-op blood work is performed. Blood samples 
will then be obtained every three months following the initiation of either adjuvant therapy or 
routine, close follow-up for up to two years. 
Pre-op consent: 
Pre-op blood draw, 4-8 week post-op blood draw, then every 3 
month blood draw following initiation of adjuvant therapy/ 
observation for up to 2 years. 
No pre-op consent: 4-8 week post-op blood draw, then every 3 month blood draw 
following initiation of adjuvant therapy/observation for up to 2 
years. 
Of note, retrospective samples will be analyzed in participants who have previously signed 
informed consent to the melanoma tissue banking protocols, DFHCC study 02-017 and 11-181.  
These participants will not need to sign the consent form accompanying this protocol. 
 
5.2 
Data Collection 
 
The sources of the data will be hospital/electronic medical records provided by the BIDMC 
Caregroup and Partners networks. Participants will be enrolled prospectively as part of the 
evaluation for the use of BRAFV600E specific inhibitors either for experimental protocols or as 
standard of care (assuming the FDA-approval of one or more of these agents) in patients with 
melanoma.  Data regarding response, progression, and survival either will be collected as part of 
the study and be made accessible to the study team or will be collected as part of routine clinical 
care in patients treated with standard of care BRAF inhibitors.   
 
The data collected will be demographic data (age, gender, vital status), coded encounter data 
(diagnoses, dates), and clinic/office notes.  The records will be reviewed electronically through 
the online medical record system at the three institutions accessed by the study team. The team 
will use a password protected computer and online medical record system maintained by the 
BIDMC, DFCI, and MGH. All study team members have HIPAA, confidentiality, and CITI 
training provided by the BIDMC, DFCI, and MGH at hiring. 
 
The database is also stored in a password protected computer at the MGH. Identifiable 
information to researchers will not be released outside the MGH study team. Unlinked 
information will be accessible through a password protected computer and database. After the 
data is retrieved from patient records, the results will be entered into the database unlinked to any 
protected health information. Any analyses, reports, or presentations of the data will not reveal 

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patient information. No risks are involved for individuals whose information will be used in this 
study. Private/confidential information will be available only to the study coordinator and the 
study team on the front sheet. 
 
5.3 
Criteria for Taking a Participant Off Study 
 
Participants will be removed from study when any of the following criteria apply: 
• Lost to follow-up 
• Withdrawal of consent for data submission 
• Death 
 
Participants will be removed from study if they request to do so. The reason for taking a 
participant off study, and the date the participant was removed, must be documented in the case 
report form (CRF). 
 
For Centralized  Subject Registrations, the research team submits a completed Off Treatment/Off 
Study form to ODQ when a participant comes off study. This form can be found on the ODQ 
website or obtained from the ODQ registration staff.  
 
For Decentralized Subject Registrations, the research team updates the relevant Off 
Treatment/Off Study information in OnCore.  
 
5.4 
Duration of Follow Up 
 
Patients will be followed until disease recurrence, death, or until closure of the study.  It is 
estimated that this study will remain open for five years.  
 
 
6. ADVERSE EVENTS  
 
6.1 
Expected Toxicities 
 
Slight transient discomfort at the puncture site as the needle is inserted is expected.  Minimal 
bleeding that can be controlled by temporary pressure and bruising at the needle site are potential 
sequelae.  
 
Toxicity Management is per phlebotomy lab standards 
 
6.2 
Adverse Event Reporting  
 
No particular adverse events are expected from participation in this protocol. The participants 
will be exposed to the minor increased risk accrued from repeated peripheral blood sampling.  
 
 

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7. MEASUREMENT OF EFFECT 
 
Not applicable in this protocol, as response to therapy will be judged per protocol specific to the 
therapy being received, which will vary from patient to patient. 
 
8. DATA REPORTING / REGULATORY REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 6.0 
(Adverse Events: List and Reporting Requirements). 
 
8.1 
Data Reporting 
 
8.1.1 Method 
 
The QACT will collect, manage, and perform quality checks on the data for this study. 
 
8.1.2 Responsibility for Data Submission 
 
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 
data forms to the QACT according to the schedule set by the QACT. 
 
8.2 
Data Safety Monitoring 
 
Involvement in this study as a participating investigator implies acceptance of potential audits 
or inspections, including source data verification, by representatives designated by the DF/HCC 
Overall Principal Investigator (or Protocol Chair) or DF/HCC. The purpose of these audits or 
inspections is to examine study-related activities and documents to determine whether these 
activities were conducted and data were recorded, analyzed, and accurately reported in 
accordance with the protocol, institutional policy, Good Clinical Practice (GCP), and any 
applicable regulatory requirements. 
All data will be monitored for timeliness of submission, completeness, and adherence to 
protocol requirements. Monitoring will begin at the time of participant registration and will 
continue during protocol performance and completion. 
8.3 
 Protocol Review and Amendments 
 
This protocol, the proposed informed consent and all forms of participant information related 
to the study (e.g., advertisements used to recruit participants) and any other necessary 
documents must be submitted, reviewed and approved by a properly constituted IRB 
governing each study location. 
Any changes made to the protocol must be submitted as amendments and must be approved 
by the IRB prior to implementation. Any changes in study conduct must be reported to the 
IRB. The DF/HCC Overall Principal Investigator (or Protocol Chair) will disseminate 

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16 
protocol amendment information to all participating investigators. 
All decisions of the IRB concerning the conduct of the study must be made in writing. 
8.4 
Ethics and Good Clinical Practice (GCP) 
 
This study is to be conducted according to the following considerations, which represent good 
and sound research practice: 
 
• E6 Good Clinical Practice: Consolidated Guidance 
www.fda.gov/downloads/RegulatoryInformation/Guidances/UCM129515.pdf 
• US Code of Federal Regulations (CFR) governing clinical study conduct and 
ethical principles that have their origin in the Declaration of Helsinki 
 Title 21 Part 11 – Electronic Records; Electronic Signatures 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr11_02.html 
 Title 21 Part 50 – Protection of Human Subjects 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr50_02.html 
 Title 21 Part 54 – Financial Disclosure by Clinical Investigators 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr54_02.html 
 Title 21 Part 56 – Institutional Review Boards 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr56_02.html 
 Title 21 Part 312 – Investigational New Drug Application 
www.access.gpo.gov/nara/cfr/waisidx_02/21cfr312_02.html 
• State Laws 
• DF/HCC research policies and procedures        
http://www.dfhcc.harvard.edu/clinical-research-support/clinical-research-unit-
cru/policies-and-procedures/ 
 
It is understood that deviations from the protocol should be avoided, except when necessary to 
eliminate an immediate hazard to a research participant. In such case, the deviation must be 
reported to the IRB according to the local reporting policy. 
 
8.5 
Study Documentation 
 
The investigator must prepare and maintain adequate and accurate case histories designed to 
record all observations and other data pertinent to the study for each research participant. This 
information enables the study to be fully documented and the study data to be subsequently 
verified. 
 
Original source documents supporting entries in the case report forms include but are not limited 
to hospital records, clinical charts, laboratory and pharmacy records, recorded data from 
automated instruments, microfiches, photographic negatives, microfilm or magnetic media, 
and/or x-rays. 
 
8.6 
Records Retention 
 

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17 
All study-related documents must be retained for the maximum period required by applicable 
federal regulations and guidelines or institutional policies. 
 
 
9. STATISTICAL CONSIDERATIONS 
 
9.1 
Study Design/Endpoints 
 
Primary Objective: To determine the sensitivity and specificity of the peripheral blood 
BRAFV600 mutation detection. 
 
Two cohorts of melanoma patients will be assessed using the blood-based assay: patients with 
advanced disease (non-resectable Stage III or Stage IV), and patients with Stage II and resectable 
Stage III disease. Assessments of diagnostic accuracy, such as sensitivity, specificity, and 
positive predictive value (PPV) of the blood-based assay, will be compared separately for the 
two cohorts, and will use paired binary data.  Sample size estimates are based on the binomial 
distribution and joint 90% confidence regions for sensitivity and specificity1,2 to adjust for the 
two comparisons being performed. Positive and negative expression will be determined using 
optimized cut points for the assay that have already been established.   
 
We are seeking evidence that the assay has the ability to detect both the presence of BRAF 
mutation in melanoma (sensitivity) and accurately indicate when a BRAF mutation is not present 
(specificity) in order to provide BRAF-directed therapy when indicated.  We propose a sample of 
110 patients with advanced melanoma to test the performance of the assay, and would require 
sensitivity of at least 0.80 and specificity of at least 0.90.  We set our target sensitivity to be 0.90 
and specificity to 0.95.  With a sample size of 110 patients (approximately 55 with BRAF 
mutation and 55 without) and the assumed target sensitivity, the lower bound of the joint 90% 
confidence region will be greater than 0.82; the lower bound of the 90% joint confidence region 
for the specificity will be approximately 0.90. 
 
We will also estimate the PPV of the assay to evaluate the practical utility of the test.  Based on a 
sample size of 110 patients, Table 1 summarizes the estimated PPV and corresponding 90% 
confidence intervals over a range of prevalence rates of BRAF mutation, using the minimum and 
target levels of sensitivity and specificity.   With a sample of 110 patients, the lower bound of the 
90% confidence interval for PPV will be no less than 0.74. 
 
Table 1 – Estimates of PPV and 90% Confidence Intervals 
Prevalence Rate 
Sensitivity 
Specificity 
PPV 
90% Confidence 
Interval for PPV 
0.4 
0.80 
0.90 
0.84 
(0.74 to 0.91) 
0.90 
0.95 
0.92 
(0.83 to 0.97) 
0.5 
0.80 
0.90 
0.89 
(0.80 to 0.94) 
                                                 
1 Alonzo TA, Pepe MS, Moskowitz CS. Sample size calculations for comparative studies of 
medical tests for detecting presence of disease.  Statist. Med 2002; 21:835-852. 
2Obuchowski NA. Sample size calculations in studies of test accuracy.Stat Methods in Medical 
Res 1998; 7: 371-392. 

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18 
0.90 
0.95 
0.95 
(0.87 to 0.98) 
0.6 
0.80 
0.90 
0.92 
(0.85 to 0.96) 
0.90 
0.95 
0.96 
(0.90 to 0.99) 
 
The cohort of patients with Stage II and resectable Stage III melanoma is an exploratory cohort. 
We cannot predict the sensitivity and specificity of the assay in patients with Stage II or resected 
Stage III disease. In addition, only patients in this cohort with T4 disease or macrometastatic 
lymph nodes will have adequate tissue for assessment.  However, we believe it is important to 
include this cohort since the ability to detect BRAF in this patient population may provide a basis 
for proposing further studies correlating BRAF detection with disease recurrence.  The 
sensitivity and specificity of the assay will be calculated and presented with 90% joint 
confidence regions.  Based upon a cohort size of 110 patients, the joint confidence regions for 
the sensitivity and specificity will be no wider than ±0.13. 
Secondary Objectives 
1. Exploration of the pharmacodynamic effects of MAPK pathway inhibitors on pre- 
and on-treatment peripheral blood BRAFV600 mutational testing. 
 
The exploration of pharmacodynamic effects will be based on the cohort of patients with 
advanced, BRAF-mutated disease confirmed by tissue analysis.  We would expect that 50% of 
the cohort would have BRAF-mutated disease and that 90% of those patients will agree to 
participate in the pharmacodynamic studies. This results in a sample of size 50.   
 
Our first investigation will correlate blood-assay results with pre-treatment radiographic 
measures of disease burden.   We hypothesize that the cases with the lowest tumor burden would 
be most likely to have a negative blood assay result.  We will use the sum of the longest diameter 
of tumor target lesions as the measure of disease burden using RECIST criteria (version 1.1). For 
this analysis, tumor burden will be a continuous outcome, and the blood assay will be classified 
as positive or negative. We will summarize baseline tumor burden descriptively and compare by 
assay result using the Wilcoxon rank-sum test.  If the blood-assay is positive in 80% of the 
samples (N=40) and negative in 20% (N=10), a Wilcoxon-rank sum test, with a two-sided type-I 
error of 10%, will have 80% power to detect a difference in disease burden that is 0.95 times the 
common standard deviation. 
 
The behavior of the blood-based assay with respect to early changes in disease will be assessed 
at the time of the first restaging.  Since there are no prior data to suggest how assay levels will 
change in response to treatment, analyses will be primarily descriptive. Two analyses are 
proposed to explore the relationship between the performance of the assay and early clinical 
response.  In both analyses, clinical response will be classified according to RECIST criteria 
(i.e., CR/PR vs. SD vs. PD vs. not assessable).  
 
At the time of the first restaging, pre- and on-treatment blood-assay levels will be compared.  
Fold-changes in assay response will be calculated (post/pre).  Fold-changes will be used as a 
continuous measure and will also be classified according to 0.5 or less (assay-based response), 2 
or greater (assay-based progression) and between 0.5 and 2 (intermediate).  Fold changes in the 
assay will be compared across RECIST responses using the Kruskal-Wallis test.  In addition, for 

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assay-based response or progression, the proportions of patients with CR/PR, SD, or PD will be 
presented with exact 90% confidence intervals.  We will further characterize the behavior of the 
assay by comparing assay fold changes with fold changes in tumor burden (post/pre); the 
relationship will be summarized graphically and using the Spearman rank correlation. 
 
Assay performance data will also be divided into four groups defined by pre-treatment 
expression and at the first restaging (i.e., +/+, +/-, -/+, -/-) and compared with response. The 
proportions will be summarized and compared using exact confidence intervals in all four assay 
combinations and also in the two subgroups with a positive assay at pre-treatment. 
 
The behavior of the blood assay over time will be summarized graphically. The status of the 
assay (+/-) will be shown at each assessment time for each patient.  Graphics will note changes in 
disease status and times of disease progression and will provide an opportunity examine patient 
follow-up for patterns of change in the assay. 
 
2. To investigate the prognostic and predictive ability of peripheral blood BRAFV600 
mutational testing in patients with Stage II or resectable Stage III melanoma. 
 
The investigation of the prognostic and predictive ability of the assay will be based on the cohort 
of patients with Stage II and resected Stage III disease. Some of these patients will have a tissue-
based BRAF assessment and a pre-treatment blood-based BRAF assay, and some of the patients 
will have the blood-based assay only.   
 
We would expect that 50% of the cohort would have BRAF-mutated disease and that 90% of 
those patients will have follow-up data from which to assess the predictive ability of the assay.  
This results in a sample of size 50.  BRAF status will be classified as positive or negative based 
on the post-resection, blood-based assay, and where applicable, for the tissue-based assessment.   
 
Prognostic Ability of the Assay 
Baseline assay results will be categorized as positive or negative and also be described on a 
continuous scale.  The proportion of patients with a positive baseline assay will be presented 
with a 90% exact binomial confidence interval. Based on a cohort size of 50, the confidence 
interval will be no wider than 0.25. Confidence intervals will also be presented for the subgroups 
of patients with intermediate- or high-risk disease.  We will also summarize continuous baseline 
assay levels using descriptive statistics for the complete cohort and according to subgroups. 
Preliminary assessments of prognostic ability will be conducted using Kaplan-Meier (K-M) 
product-limit estimates for relapse or survival and stratified log-rank tests. K-M estimates will be 
stratified by AJCC stage and assay results to explore assay performance across different 
prognostic groups.   Relapse-free survival and overall survival estimates at pre-specified time 
points will be presented with 90% confidence intervals estimated using log(-log(survival)) 
methodology.  A secondary analysis will be conducted in the cohort with blood- and tissue-based 
assessments according to the four groups defined by their combined positive/negative status to 
look for differences in prognosis. 
 

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The relative effect of the blood-based assay with respect to relapse or survival will be estimated 
using Cox regression models.  Since the AJCC staging incorporates prognostic factors such as 
number of involved nodes, tumor burden, ulceration, and tumor thickness, the Cox models will 
be stratified by AJCC stage to allow for differences in the baseline hazard of relapse or death.  
Models will include as covariates age, site of the primary melanoma, sex, and baseline serum 
LDH in addition to the blood-assay results. We will use aggregated data for the primary models; 
secondary models will be fit separately for the intermediate- and higher-risk cohorts.  Hazard 
ratios for the assay will be presented with 90% confidence intervals. 
 
Predictive Ability of the Assay 
 
The behavior of the blood-based assay over time will be summarized graphically by AJCC stage 
or according to melanoma treatment.  The status of the assay will be shown at each assessment 
time for each patient, in addition to time of disease progression. Of particular note will be 
assessment times where a previously undetectable assay becomes detectable, or times where 
there is a doubling in the assay relative to baseline. Treatment will be classified into four groups: 
immunotherapy, BRAF/MEK inhibitors, other translational signal transduction/targeted 
therapies, or chemotherapy (cytotoxic therapy).   
The first assessment of assay performance over time will take place at 3 months and will be 
based on fold-changes in the quantified assay relative to baseline.  This early look will minimize 
the number of patients lost due to rapid progression of disease.  Based on 18-month progression 
rates reported in the literature, we estimate that approximately 11% of patients will have 
progressed by the time of the 3-month assessment.  For an initial cohort size of 50 patients, and 
assuming that 45 patients will have 3-month assay, a Wilcoxon-signed-rank test, comparing the 
observed fold-change with one, with a two-sided type-I error of 10%, will have 80% power to 
detect a difference in fold-change that is 0.41 times the standard deviation. 
In more exploratory analyses, we will model the behavior of the assay over time using repeated 
measures, random-effects regression with the continuous assay measure as the dependent 
variable.  Models will include as covariates AJCC stage and treatment classification, in addition 
to other prognostic factors.  To investigate the relationship between the assay and PFS (or OS), 
we will use the extended Cox (EC) model with assay performance as the time-dependent 
covariate. Models will be stratified by AJCC stage, and will include treatment group and 
prognostic factors as predictors. In separate EC models, the assay will be modeled (a) on a 
continuous scale, (b) based on fold-changes from baseline, and (c) based on an indicator of assay 
response (double the baseline level or change from non-detectable to detectable).             
 
Characteristics of Study Sample 
 
Patient demographic and disease characteristics will be presented in the aggregate and by cohort.  
Characteristics measured on a continuous scale, such as age, will be summarized using N, mean, 
standard deviation, median and range.  Categorical characteristics will be summarized using N 
and percentage. 
 
 

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21 
10. PUBLICATION PLAN 
 
The results should be made public within 24 months of reaching the end of the study. The end of 
the study is the time point at which the last data items are to be reported, or after the outcome 
data are sufficiently mature for analysis, as defined in the section on Sample Size, Accrual Rate 
and Study Duration. If a report is planned to be published in a peer-reviewed journal, then that 
initial release may be an abstract that meets the requirements of the International Committee of 
Medical Journal Editors. A full report of the outcomes should be made public no later than three 
(3) years after the end of the study.  

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