Fluorescence-Guided Detection of Malignant Gliomas: A Dose Ranging Study Using 5-Aminolevulinic Acid (ALA) Induced Protoporphyrin (PpIX) in a Multicenter Phase II Clinical Trial
Phase: PHASE2
Conditions: Brain and Central Nervous System Tumors
Sponsor: Andrew Sloan, MD

 
CASE 1305 Protocol Version dated 09.15.17 Page 1 of 30 
 
Case Comprehensive Cancer Center 
 
STUDY NO: 
CASE 1305 
 
TITLE:  
FLUORESCENCE-GUIDED DETECTION OF MALIGNANT GLIOMAS: A DOSE 
RANGING STUDY USING 5-AMINOLEVULINIC ACID (ALA) INDUCED 
PROTOPORPHYRIN IX (PpIX) IN A PHASE II CLINICAL TRIAL. 
 
PRINCIPAL INVESTIGATOR: 
 
Andrew Sloan, MD 
 
Department of Neurological Surgery 
University Hospitals of Cleveland 
Case Western Reserve University 
11100 Euclid Avenue 
Cleveland, OH 44106-5042 
 (216) 844- 6054 
 
CO-INVESTIGATORS:  
Listed on next page 
 
SPONSOR: 
 
 
CASE COMPREHENSIVE CANCER CENTER 
 
CLINICAL FACILITIES:  
Seidman Cancer Center, University Hospitals of Cleveland 
Cleveland, OH 
 
APPROVALS: 
 
 
Protocol Review and Monitoring Committee Approval:  10/31/2005 
 
 
 
 
UH-CMC/IRB Approval: 1/17/2006 
 
 
 
 
GCRC Approval: 5/3/06 (now DCRC) 
 
STUDY COORDINATORS: 
Listed on next page. 

 
CASE 1305 Protocol Version dated 09.15.17 Page 2 of 30 
 
Case Comprehensive Cancer Center 
 
 
CWRU CO-INVESTIGATORS: 
 
                                             
 
 
 
Warren Selman, M.D.  
                  
Department of Neurological Surgery 
 
University Hospitals of Cleveland 
 
Case Western Reserve University 
 
11100 Euclid Avenue  
 
 
Cleveland, OH 44106  
 
 
216-844-5745 
 
 
  
 
Mark Cohen, M.D. 
 
 
         
Department of Pathology                                        
 
University Hospitals of Cleveland                         
 
Case Western Reserve University 
       
11100 Euclid Avenue  
 
       
Cleveland, OH 44106  
 
       
 
216-844-8928 
                                          
 
 
 
 
STUDY COORDINATORS: 
 
Christopher Murphy, RN 
SCC 
Case Western Reserve University 
 
11100 Euclid Avenue  
 
 
Cleveland, OH 44106  
 
 
 
BIOSTATISTICIANS 
Jill S. Barnholtz-Sloan, PhD 
 
 
Abdus Sattar, PhD 
Case Comprehensive Cancer Center 
 
Case Comprehensive Cancer Center 
11100 Euclid Ave, Wearn 152 
 
 
11100 Euclid Ave, Wearn 152 
Cleveland, Ohio 44106 
 
 
Cleveland, Ohio 44106 
216-368-1506 
 
 
 
216-368-1501 
 
NON-CWRU INVESTIGATORS: 
 
Brian Wilson, Ph.D. 
Div. of Biophysics & Bioimaging 
University Health Network 
610 University Ave., 7416 
Toronto,  M52M9 Canada 
Wilson@uhmres.utoronto.ca 
416 946-2952 

 
CASE 1305 Protocol Version dated 09.15.17 Page 3 of 30 
 
TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
 
 
 
PAGE 
 
SCHEMA 
 
 
 
 
 
 
 
 
5 
 
1.0 
INTRODUCTION [background and significance] 
 
 
 
6-7 
 
2.0 
OBJECTIVES 
 
 
 
 
 
 
 
7 
2.1 
PRIMARY AIM  
 
 
 
 
 
7 
2.2 
PRIMARY OBJECTIVE  
 
 
 
 
7 
2.3 
SECONDARY OBJECTIVE 
 
 
 
 
7 
2.4 
TERTIARY OBJECTIVE  
 
 
 
 
7 
 
3.0 
ELIGIBILITY CRITERIA 
 
 
 
 
 
8-9 
 
3.1 
PATIENT ELIGIBILITY CRITERIA 
 
 
 
8 
 
3.2 
EXCLUSION CRITERIA  
 
 
 
 
8-9 
 
4..0 
RESEARCH DESIGN 
 
 
 
 
 
 
9-17 
 
4.1 
SUMMARY OF TRIAL DESIGN  
 
 
 
9 
 
4.2 
RANDOMIZATION   
 
 
 
 
 
9-10 
 
4.3         DSMB and DSCTC                          
 
 
 
10 
 
 
4.3.1 
DATA SAFETY MONITORING BOARD    
 
10 
 
 
4.3.2 
DATA SAFETY TOXICITY COMMITTEE    
 
10 
 
 
 
 
 
 
 
4.3.3 
SITES FOR SURGICAL RESECTION AND 
 
10 
 
 
 
FLUORESCENCE IMAGING 
 
 
4.3.4 
SITES FOR TISSUE ANALYSIS  
 
 
10-11 
 
4.4 
PRE-OPERATIVE PROCEDURES 
 
 
 
11 
 
 
4.4.1 
PATIENT INFORMED CONSENT 
 
 
11 
 
 
4.4.2 
LIGHT SENSITIVITY PRECAUTIONS 
 
 
11 
4.4.3 
PATIENT GROUPS AND NUMBERS, ALA DOSE  
11 
 
4.4 
SURGICAL PROCEDURES 
 
 
 
 
12-13 
4.4.1 
PRECAUTIONS FOR PHOTOSENSITIVITY DURING 
12 
SURGERY 
 
 
4.4.2 
SURGICAL TREATMENT 
 
 
 
12 
 
 
4.4.3 
INTRA-OPERATIVE MEASUREMENTS  
 
12 
 
 
 
4.4.3.1 BULK TUMOR MEASUREMENTS 
 
12-13 
 
 
4.4.4 
SPECIMEN HANDLING  
 
 
 
13 
 
 
 
 
 
4.5 
TISSUE AND DATA ANALYSIS  
 
 
 
13-16 
 
 
4.5.1 
TISSUE ANALYSIS 
 
 
 
 
13-14 
 
 
 
4.5.1.1 1ST PART OF BIOPSY:   
 
 
13 
HISTOPATHOLOGICAL ANALYSIS 
 
 
 
4.5.1.2 2ND PART OF BIOPSY: PpIX QUANTIFICATION 13 
 
 
 
4.5.1.3 3RD PART OF BIOPSY: (CONFOCAL) 
 
14 
 
 
 
 
FLOURESCENCE MICROSCOPY 
4.6 
POST OPERATIVE ACTIVITIES  
 
 
 
14 
 
 
4.6.1 
POST OPERATIVE BLOOD SAMPLE COLLECTION 
14 
 
4.7 
REPORT ADVERSE EVENTS ASSOCIATED 
 
 
14-17 
 
 
 
WITH SYSTEMIC ALA 
 
 
4.7.1 
SKIN PHOTOSENSITIVITY 
 
 
 
14-15 
 
 
4.7.2 
NAUSEA AND VOMITING 
 
 
 
15 
 
 
4.7.3 
LIVER FUNCTION TEST ABNORMALITIES 
 
15 

 
CASE 1305 Protocol Version dated 09.15.17 Page 4 of 30 
 
 
 
4.7.4 
OTHER EVENTS 
 
 
 
15 
 
4.8 
ADVERSE EVENT REPORTING 
 
 
 
15-17 
 
5.0 
MEASUREMENT OF EFFECT 
 
 
 
 
 
17-18 
5.1 
ENPOINTS 
 
 
 
 
 
 
17 
 
5.2  
DATA ANALYSIS 
 
 
 
 
 
17 
 
 
5.2.1 
FLUORESCENCE INTENSITY LEVELS VS. 
 
17 
 
 
 
HISTOPATHOLOGY 
 
 
5.2.2 
FLUORESCENCE LEVELS VS. PpIX EXTRACTION 
17 
 
 
 
ASSAY 
 
 
5.2.3 
CONFOCAL FLUORESCENCE MICROSCOPY VS. 
18 
 
 
 
HISTOPATHOLOGY 
 
6.0 
STATISTICAL PARAMETERS 
 
 
 
 
 
18 
6.1 
PATIENT DATA SET 
 
 
 
 
 
18 
 
7.0 
DRUG FORMULATION AND PROCUREMENT 
 
 
 
18-19 
 
7.1 
ALA FORMULATION AND TOXICITIES  
 
 
18 
 
 
7.1.1 
ALA FORMULA 
 
 
 
 
18-19 
7.1.2 
ALA PREPARATION 
 
 
 
 
19 
 
7.2 
ALA SUPPLY 
 
 
 
 
 
 
19 
 
7.3 
ALA STORAGE  
 
 
 
 
 
19 
 
7.4 
ALA PROCUREMENT  
 
 
 
 
19 
 
8.0 
STATISTICAL CONSIDERATIONS 
 
 
 
 
19-22 
8.1 
STATISTICAL ANALYSIS PLAN 
 
 
 
19 
8.2 
SAMPLE SIZE AND STATISTICAL POWER 
 
 
20 
 
9.0  
PLAN FOR INCLUSION OF VULNERABLE POPULATIONS 
 
23 
 
9.1 PLAN FOR INCLUSION OF ILLITERATE INDIVIDUALS 
 
23 
 
9.2 PLAN FOR INCLUSION OF NON-ENGLISH SPEAKING INDIVIDUALS 23 
 
9.3 PLAN FOR INCLUSION OF PREGNANT WOMEN AND FETUSES 
 23 
 
 
 
 
 
 
 
 
 
10.0 
RECORDS TO BE KEPT  
 
 
 
 
23-24 
 
11.1 Electronic Medical Records 
 
 
 
24 
 
11.0 
LITERATURE CITED 
 
 
 
 
 
 
24-27 
 
APPENDICES:   
 
 
 
 
 
 
 
28-30 
 
12.0       APPENDIX I: SKIN PHOTOSENSITIVY DUE TO ALA 
 
 
28 
              APPENDIX II: DESCRIPTION OF THE FLUORESCENCE IMAGING SYSTEM 29 
              APPENDIX III: STUDY FLOW CHART                                                                     30 
 
 
 
 
 
 
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 5 of 30 
 
SCHEMA:   
 
Two arm randomized trial of newly diagnosed or recurrent supratentorial malignant 
astrocytic tumors. 
 
 
NEWLY DIAGNOSED OR RECURRENT MALIGNANT 
ASTROCYTIC TUMORS: IDENTIFICATION AND CONSENT 
 
 
 
 
RANDOMIZATION 
 
 
 
 
ADMINISTRATION OF ALA 
 
 
 
 
SURGICAL RESECTION: 6 BIOPSIES FROM 3 FLUORESCENT REGIONS 
 (i.e., 2 bx from each of 3 sites) 
 
 
 
 
ASSESSMENT OF CORRELATION BETWEEN SUBJECTIVE AND OBJECTIVE 
FLUORESCENCE INTENSITY, 
DRUG DOSE,  
AND % TUMOR/ % NORMAL TISSUE PRESENT IN BIOPSY,  
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 6 of 30 
 
1.0  
INTRODUCTION 
 
 
The treatment of patients with high grade gliomas remains a challenge for modern 
therapy. The prognosis for these patients is poor; the median survival time after diagnosis 
and treatment is less than 1 year [1, 2] and may be linked to the completeness of tumor 
removal. [3-6]. A recent study of 416 patients with glioblastoma multiforme (GBM) 
indicates that resection of 89% of the tumor volume is necessary to improve survival and 
a significant survival advantage of 4.2 months was associated with resection of 98% or 
more of the tumor volume, compared with resection of less than 98% [7]. However, the 
highest possible degree of tumor resection is often limited by the surgeon’s ability to 
distinguish residual tumor tissue from surrounding brain tissue under conventional white 
light surgical microscope illumination [8]. For these reasons, methods enabling better 
intraoperative discrimination of viable tumor borders may be valuable. 
 
Fluorescence imaging may, potentially, enhance the contrast of viable tumor borders. 
Although administration of fluorescent markers to enhance contrast of malignant gliomas 
is not new [9, 10, 11], marking tumors with 5-amino levulinic acid (ALA) is conceptually 
different from other investigations.  ALA is not itself fluorescent but is metabolized into 
the strongly fluorescent proto-porphyrin IX (PpIX) by a number of malignant tumors in 
situ through enzymes of the heme-biosynthesis pathway [12]. This may avoid pitfalls 
when a fluorescent marker is administered directly, such as leakage from the tumor into 
surrounding normal brain tissue [13].  
 
ALA-PpIX fluorescence imaging has previously been investigated at St. Michael's 
Hospital and the University Health Network, Toronto Cancer Institute [Drs. N. Marcon 
and B. Wilson]. These studies examined the utility of ALA for detection and 
photodynamic therapy (PDT) of dysplasia in patients with Barrett’s disease of the 
esophagus.  
 
Recently, ALA-PpIX fluorescence imaging was introduced by a German group for 
intraoperative detection and resection of residual malignant glioma [14, 15]. In the latter 
study [15], 52 patients were investigated using an operating room-adapted microscope for 
fluorescence imaging. ALA was administered orally at a dose of 20 mg/kg 3 hours before 
induction of anesthesia. These workers found that leaving “solidly fluorescing” tissue 
unresected had a negative influence on survival, whereas the prognosis of patients with 
residual “vague fluorescence” was not significantly different from that of patients in 
whom all fluorescent tissue was removed completely. Histologically, solidly fluorescing 
tissue was mostly characterized by coalescent tumor cells, whereas vaguely fluorescing 
tissue usually represented infiltrating tumor of intermediate or low cellular density. 
Analysis of tumor biopsy specimens revealed infiltrating tumor also to be present beyond 
vaguely fluorescing tissue portions. Recent data suggest that the sensitivity of this dose 
and administration timing for 5-ALA is 75% while specificity is only 71% [16].  While 
encouraging, this study may be considered subjective as it involved only one 
neurosurgeon and was not quantitative in determining the levels of PpIX fluorescence in 
the tumor and normal tissue.  The definitions of “solidly fluorescing…” and “vaguely 
fluorescining are unclear and imprecise.  In addition, all published studies have been 
performed using the 20 mg/kg 5-ALA dose and 3 hr pre-operative administration.   

 
CASE 1305 Protocol Version dated 09.15.17 Page 7 of 30 
 
Unpublished reports suggest 2 mg/kg was not detectable (Stummer personal 
communication),_but values between 2 and 20 mg/kg from a clinical trial setting have not 
been published. 
 
Our group is developing point-probe systems for intra-operative quantitative PpIX 
concentration measurement. A 1st generation instrument has been developed and used in 
another clinical trial [16, 17]. 
 
2.0 
OBJECTIVES 
 
2.1 
PRIMARY AIM  
 
 
Our primary long-term goal is to improve the completeness of surgical resection of 
malignant brain tumor through image- guided fluorescence localization. We hypothesize 
that the use of qualitative fluorescence imaging and point PpIX concentration 
quantification will enable more complete tumor resection than normal direct (i.e., white 
light) visualization, and thereby improve patient survival [7].  
2.2 
PRIMARY OBJECTIVE 
Primary objective: As a first step, we will assess 2 doses of 5-ALA, 10mg/kg and 20 
mg/kg, to determine the optimum ALA dose in terms of both sensitivity and specificity 
for residual tumor.  We will compare residual tumor detection by both in vivo qualitative 
and quantitative fluorescence imaging using histology of the biopsied tissue as the gold 
standard. 
2.3  
SECONDARY OBJECTIVE 
Secondary Objective: We will assess the correlatation between the recorded in vivo 
qualitative assessment of fluorescence signal from the neurosurgeon with the post-
surgical (i.e., ex vivo) absolute PpIX concentration detected both intraoperatively and in 
ex vivo tissue biopsies.  
 
2.4  
TERTIARY OBJECTIVE 
Tertiary Objective: To determine the association between the presence of fluorescence in 
the surgical cavity and the post-operative image enhancement on MRI. This includes the 
relationship between the amount and location of residual tumor detected by fluorescence, 
PpIX concentration, and intra-operative frameless stereotaxy following maximal 
resection versus the amount and location of tumor imaged post-operatively via CT and/or 
MRI. 
 
 
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 8 of 30 
 
3.0 
ELIGIBILITY CRITERIA 
 
3.1 
PATIENT ELIGIBILITY CRITERIA 
 
 
Patients will be eligible for this study if they meet all of the following criteria: 
 
Table 1: Inclusion Criteria 
Age 
18 years or older 
Tumor Pathology 
Suspected or confirmed newly diagnosed or recurrent malignant 
gliomas WHO grade IV 
Location 
Supratentorial 
Resection 
Tumor must be judged suitable for resection on the basis of 
imaging studies. 
Consent 
Patients must be able to give written, informed consent as 
approved by the local IRB 
Newly Diagnosed Tumors 
Patients with newly diagnosed suspected Grade IV glioma  who 
have had not been previously treated with cranial radiation 
therapy 
Recurrent Tumors 
Patients with recurrent Grade IV gliomas  who have failed 
cranial radiation therapy 
 
It is the policy of the Case Comprehensive Cancer Center to strive for gender and 
minority patient participation that represents the population of Northeast Ohio in all 
clinical investigations. Both men and women of all races and ethnic groups are eligible 
for this trial.   
 
Grade IV gliomas will likely have a different PpIX uptake and thus fluoresce differently 
compared to Grade III gliomas [15] therefore, this study will only focus on Grade IV 
gliomas. However, the grade of the glioma is not certain until surgical resection of the 
glioma at which point biopsies are taken for histopathological determination of the 
glioma grade. As a result some patients with Grade III gliomas may be enrolled in this 
study, but these patients will be excluded from primary analysis. The data obtained from 
patients with Grade III gliomas will be used to explore differences in fluorescence 
between Grade III and Grade IV brain tumors.   We do not expect that this will result in a 
statistically significant difference, but only that generate hypothesis for future studies. 
 
3.2 
EXCLUSION CRITERIA 
 
 
Patients will be excluded from participation in the study if they meet any of the following 
criteria: 

 
CASE 1305 Protocol Version dated 09.15.17 Page 9 of 30 
 
Table 2: Exclusion Criteria 
Pregnant women or those who are breast feeding 
Individuals with history of cutaneous photosensitivity, porphyria, hypersensitivity to 
porphyrins, photodermatosis, exfoliative dermatitis 
Individuals with history of liver disease in last 12 months 
Individuals with AST, ALT, ALP, or bilirubin >2.5x normal upper limit any time during the 
previous 2 months 
Individuals with plasma creatinine>180 μmol/L 
Individuals who are unable to comply with photosensitivity precautions 
Individuals without a probable or expected grade IV glioma 
 
A baseline blood sample will be drawn for creatinine, AST, ALT, ALP, and bilirubin to 
verify whether or not this exclusion criterion is met. 
 
 
4.0 
RESEARCH DESIGN 
 
4.1 
SUMMARY OF TRIAL DESIGN 
Summary of Trial Design: This study will enroll evaluable patients undergoing surgical 
resection of malignant, grade IV gliomas in both of two groups:  those with newly 
diagnosed GBM and those with recurrent GBM.   Patient, in each group (primary vs 
recurrent GBM)  will be randomized to one of 2 levels of ALA dose (10 and20 mg/kg) to 
be given orally at 3 hours prior to anticipated midpoint of surgery.  
 
4.2  
RANDOMIZATION 
 
Patients who have consented to this protocol will be randomly assigned to one of two 
ALA dose groups (Table 3). Randomization will be stratified by whether the tumor is 
newly diagnosed (i.e. de novo) or recurrent. The data center will prepare sealed, opaque 
envelopes with the randomized assignment to ALA dose and administration time and 
notify the pharmacy of the trial site so that so that the correct ALA dose can be prepared. 
Neurosurgeons and investigators responsible for determining pathologic characteristics, 
for quantifying fluorescence images, for extracting chemical PpIX,  for co-localizing 
fluorescence images with MR images will be blinded to the ALA dose and administration 
time. In case of emergency, such as toxicity or allergic reaction attributable to the drug, 
the research pharmacist on call will break the blind and inform the PI and the physician 
responsible for clinical management.  
 
A safety monitoring board has been developed at the Case Comprehensive Cancer Center 
at Case Western Reserve University. Also, forms will be filled out by the surgeon 
postoperatively on a daily basis, the patient’s surgeon will report any adverse effects. In 
addition to specific signs and symptoms, liver function tests will be ascertained at 48 

 
CASE 1305 Protocol Version dated 09.15.17 Page 10 of 30 
 
hours and, if abnormal, these tests will be continued on a twice-weekly basis until they 
peak and then at weekly intervals thereafter to resolution. In the case of any adverse 
event, the patient will be examined by the study physician and appropriate medical action 
taken. 
 
4.3  
DSMB AND DSTC 
4.3.1. Data Safety Monitoring Board (DSMB) 
The Data Safety Monitoring Board for this study is: 
 
The Standing Data Safety Monitoring Board of the Case Comprehensive Cancer Center, 
Case Western Reserve University, School of Medicine. 
 
4.3.2.  Data Safety Toxicity Committee (DSTC) 
Case Comprehensive Cancer Center Data Safety and Toxicity Committee 
 
Anjali Advani, M.D., Chair, DSTC 
Paolo Caimi, M.D., Co-Chair, DSTC 
 Katarzyna Karelus., Administrative Director Case Comprehensive Cancer Center 
Clinical Research Office 
 
In addition, the Case Comprehensive Cancer Center Data and Safety Toxicity Committee 
will review all serious adverse events and toxicity reports as well as annual reviews. 
4.3.3 Site for surgical resection and fluorescence imaging  
Surgical resection and fluorescence imaging will be performed by neurosurgeons at 
University Hospitals Cleveland Medical Center. 
 
Image processing for quantification of the fluorescence signal and clinical support, 
training and maintenance for the fluorescence imaging devices will be provided by Dr. 
Wilson’s lab. 
 
In addition, a dedicated person will be assigned that is responsible for on-site clinical 
support, data/sample management, data/sample transfer to Dr. Wilson’s lab, and on-site 
maintenance and storage of the fluorescent imaging device. 
4.3.4 Sites for tissue analysis 
Tissue samples will be sent to the study neurohistopathologist for assessment: 
Mark Cohen, MD, Neuropathologist Department of Pathology 
University Hospitals of Cleveland 
Case Western Reserve University 
 
 
 
11100 Euclid Avenue 
 
 
Cleveland, OH 44106 
 
216-844-8928  
 
Frozen tissue samples will be sent to Dr. Wilson’s lab for fluorescence microscopy and 
PpIX extraction. 

 
CASE 1305 Protocol Version dated 09.15.17 Page 11 of 30 
 
 
Brian Wilson, Ph.D. 
Div. of Biophysics & Bioimaging 
University Health Network 
610 University Ave., 7416 
Toronto,  M52M9 Canada 
Wilson@uhmres.utoronto.ca 
416 946-2952 
 
4.4 
PRE-OPERATIVE PROCEDURES 
 
4.4.1 PATIENT INFORMED CONSENT 
 
Patients will enter this study on a voluntary basis. Enrolled patients receive an 
information package that includes an informed consent form. This package will inform 
the patient about the objectives of the study, the procedures that will be used, the 
potential risks involved, and the procedures that they should undertake to avoid possible 
photosensitivity reactions. At the time of obtaining consent for surgery, the attending 
surgeon or their delegate will obtain informed consent for this trial. 
 
 
4.4.2 LIGHT SENSITIVITY PRECAUTIONS 
 
Before ALA administration, patients will be given verbal and written instructions for 
protection from sunlight by the staff surgeon or his delegate (see Section 4.7.1). It should 
be noted that these patients are hospitalized following surgery, so that there is in practice 
a very small possibility of their being exposed to bright sunlight outdoors. Nursing staff 
in the recovery room and on the ward will be instructed regarding the need to avoid 
accidental exposure to bright light in the first 24 hours. 
4.4.3 .PATIENT GROUPS AND NUMBERS AND ALA DOSE 
 
This clinical trial has ALA dose at 2 levels (10 and 20 mg/kg) and ALA administration 
time at 1 time point (3h). Consenting patients scheduled for surgery will be assigned to 
primary or recurrent arm based on clinical presentation, are then randomly assigned to 
one of two groups (for dose assignment) within   each of the two arms of the study. 
Sample sizes of the 4 patient groups with two ALA dose levels are listed in the table 
below: 
 
Table 3: Study Design and Sample Size 
 
Administration Time 
ALA dose 
3 hours 
New Dx 
3 hours 
Recurrent Dx 
 
 
 
10 mg/kg 
30 
30 
20 mg/kg 
30 
30 
For both patients with new and recurrent disease, biopsies will be taken at up to six sites 
identified by the surgeon:  in the tumor center, tumor edge, area surrounding the tumor (if 
safe), areas seen to fluoresce intraoperatively and an area with MR enhancement outside 

 
CASE 1305 Protocol Version dated 09.15.17 Page 12 of 30 
 
the tumor region (if this can be accomplished safely). Prior to collecting these biopsies 
readings will be taken at the biopsied location with the PpIX point probe by the surgeon. 
For each of the 6 biopsies, they will be divided into 3 parts and distributed for further 
analysis as follows: one portion will be sent to the pathologist for assessment of tumor 
percentage, one portion will be evaluated by the Division of Biophysics at the University 
of Toronto for PpIX concentration and the other for assessment of fluorescence. 
 
4.4 
SURGICAL PROCEDURES 
 
4.4.1  PRECAUTIONS FOR PHOTOSENSITIVITY DURING SURGERY 
 
During surgery, light-opaque drapes will be used to cover all skin, including the area 
around the surgical site. The patient’s eye lids will be closed with tape and covered with 
goggles that block the appropriate light wavelengths that might damage the retina.  The 
patient will receive instructions on how to prevent exposure to sunlight and bright indoor 
lighting for a period of 24 hours post surgery (see Appendix I: Section 12). 
4.4.2 SURGICAL TREATMENT 
 
During surgery, the intraoperative fluorescence observations and PpIX concentration 
measurements will be taken by the surgeon and recorded by research personalle. The 
surgical site will be illuminated with UV/blue light in the wavelength range of 350-
490nm with an irradiance of 20-40 mWcm-2. A fluorescence image will be taken using a 
commercially available operating room microscope. A white-light image will also be 
taken using the same microscope in non-fluorescence mode. Each tissue biopsy will be 
taken at the discretion of the operating surgeon (the biopsy location on the fluorescence 
image will be recorded) and divided in 3 parts that will be analyzed by histopathology, 
fluorometry, and fluorescence microscopy. At each site, the position of the biopsy in the 
tumor bed will be determined by frameless stereotaxy using an Optical Tracking System 
(OTS) for correlation with pre- and post-operative CT and/or MRI scans. Biopsies of 
50mg  or less are taken with biopsy forceps .  
4.4.3 INTRA-OPERATIVE MEASUREMENTS 
 
The procedures for performing fluorescence imaging and spectroscopy during and 
following resection are well established from our prior experience. 
4.4.3.1  
BULK TUMOR MEASUREMENTS 
 
1. Under white light illumination, the surgeon will identify biopsy sites of 
tumor and normal tissue with a probe and corresponding white-light and 
fluorescence images will be taken.  
2. A biopsy will be taken of each of the sites identified under white light 
illumination and under fluorescence imaging. 
 
Additionally, the location of the biopsies will be determined with an Optical Tracking 
System for post-operative correlation with pre- and post-operative CT and/or MR-scans.     
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 13 of 30 
 
The exact step-by step operating room procedures for these intra-operative measurements 
are described in Appendix III, Section 12. 
 
4.4.4  
SPECIMEN HANDLING 
 
Each biopsy will be divided immediately by the pathologist using a scalpel into 3 parts: 1 
will be placed in formalin for standard histopathological assessment. The other 2 parts 
will be protected from light exposure, snap frozen in liquid nitrogen and will be kept at -
80 ºC in a light protected environment until further use, that is analysis by 
spectrofluorimetry and confocal fluorescence microscopy (see below for procedures). 
 
The intra-operative fluorescence imaging information will not be used in this trial to 
modify or extend the surgical resection. 
 
4.5 
TISSUE AND DATA ANALYSIS 
 
4.5.1 TISSUE ANALYSIS 
 
Each single biopsy, taken for the purposes of this single-center study, is divided in 3 parts 
to perform 3 different types of analyses. All of these tissue samples will be analyzed 
according to standard operating procedures for each analysis.  . These biopsies will not be 
used as part of patient care.  
 
Note: In case a biopsy is too small to be divided in three parts, the biopsy will be fixed in 
formalin and given to Dr. Cohen for histopathological analysis only. 
 
4.5.1.1  
1ST PART OF BIOPSY: HISTOPATHOLOGICAL ANALYSIS 
 
The first part of each biopsy will be fixed in formalin and given to Dr. Cohen who will 
prepare standard Hematoxylin & Eosin (H&E) stained sections. This histopathology will 
be assessed by a single experienced neuropathologist, Dr. Cohen. Grading and 
assessment of tumor extent will be determined in the usual fashion.  
 
4.5.1.2  
2ND PART OF BIOPSY: PpIX QUANTIFICATION 
 
The second part of each biopsy will be protected from light exposure, immediately snap-
frozen in liquid nitrogen, and stored at -80 ºC before being sent to Dr. Wilson’s 
laboratory at the University Health Network Hospital in Toronto. The PpIX concentration 
of that sample will be determined using a procedure developed by Lilge et al. [19] which, 
briefly, is as follows: After thawing, the tissue will first be finely chopped with a scalpel 
blade, and suspended by placing it in SolvableTM (Dupont, Boston) at 50 C in a water 
bath and shaken for 1 hour, then mechanically homogenized and re-incubated in Solvable 
for another hour. The fluorescence of aliquots of the suspended sample will then be 
measured in a scanning spectrofluorimeter (PTI, London, ON, Canada). The PpIX 
concentration will then be determined by spiking the sample with known concentrations 
of PpIX. This procedure is currently used as the standard technique in the same 

 
CASE 1305 Protocol Version dated 09.15.17 Page 14 of 30 
 
investigator-group’s NIH-sponsored, multi-center, Photofrin-mediated PDT brain tumor 
clinical trial.  For 100 mg brain tissue samples, it has a lower detection limit of 0.02g 
PpIX per gram of tissue. The anticipated concentration range in tumor for the lowest 
ALA dose is expected to be 100 times this value. 
4.5.1.3  
3RD PART OF BIOPSY: (CONFOCAL) FLUORESCENCE 
MICROSCOPY 
 
The third part of each biopsy will be protected from light exposure and immediately 
snap-frozen in liquid nitrogen. It will then be light protected and stored at -80 ºC in to Dr. 
Wilson’s lab in Toronto. (Confocal) fluorescence microscopy will be used to determine 
the microdistribution of PpIX. This technique is also in routine use in Dr. Wilson’s 
laboratory [17]. Briefly, serial 5m frozen sections of each biopsy will be made. 
Alternate sections will be stained for standard histological comparison with the H&E 
sections above. The quality of H&E sections made in this way is not adequate for clinical 
diagnosis, but does allow direct one-to-one identification of areas of tumor involvement 
when compared with the confocal fluorescence microscopy of the adjacent sections. The 
fluorescence images will be obtained using a UV/visible Zeiss (Thornwood, NY) 510 
confocal fluorescence microscope equipped with multiple detection wavelength channels. 
Regions of tumor and normal brain tissue will be identified and the relative fluorescence 
intensity and heterogeneity of the fluorescence will be quantified.  
 
 
4.6 
POST OPERATIVE ACTIVITIES 
 
 
4.6.1 POST OPERATIVE BLOOD SAMPLE COLLECTION 
 
Blood will be drawn for liver function tests 48 hours post tumor resection. If abnormal (> 
than Grade 3), weekly liver function tests will be drawn for 2 weeks. They will then be 
monitored on a monthly basis until they normalize (see Section 4.7.3). Subsequent to 
study completion, patients will have regular surveillance as per the current standard of 
care. 
 
4.7 
REPORTING ADVERSE EVENTS ASSOCIATED WITH SYSTEMIC 
ALA 
 
There have been over 19 published reports of systemic administration of ALA for PDT in 
at least 230 patients [14, 15, 20-40] with ALA doses up to 60 mg/kg. No serious adverse 
events or reactions have been reported; however, skin photosensitivity, nausea and 
vomiting, liver function abnormalities and some other events (as outlined below have 
been temporally related to ALA administration). 
 
4.7.1 SKIN PHOTOSENSITIVITY 
 
Since ALA-induced PpIX accumulation occurs mostly in cells of epithelial origin, 
accumulation in skin and subsequent cutaneous injury after sun exposure has been 
reported.  Four volunteers, who ingested doses of ALA ranging from 12.5 to 35 mg/kg, 
developed photosensitization of skin (but no sunburns) that persisted for approximately 
24 hours [37], and a severe sunburn occurred 8 hours after a 10 mg/kg load in a volunteer 
who was exposed to bright light [38].  The reactions reported in clinical PDT trials (30-60 

 
CASE 1305 Protocol Version dated 09.15.17 Page 15 of 30 
 
mg/kg) were usually mild, consisted mainly of erythema or a “mild photosensitivity 
reaction” and resolved within 24 to 48 hours.  Blister formation and skin necrosis has not 
been reported [40]. Ocular sensitivity has not been reported with systemic ALA. 
 
Based on this evidence, the patients in our study will receive written and verbal 
instructions regarding skin exposure to ambient light (see Appendix I).  They will be 
advised to stay indoors for 1 day (24 hours), cover exposed parts of their bodies, and 
protect their eyes from direct sun rays, strong fluorescent or incandescent lighting (e.g. a 
dentist's lamp or examining light) or strong residential direct indoor lighting (e.g. direct 
sunlight through a window for more than for more than a few minutes, direct spotlights, 
floodlights, etc.) for this period.  After 1 day, they may expose a small area of skin to the 
sun for 15 minutes to test for residual sun sensitivity.  There is no proof that sun screens 
are of any value. 
 
4.7.2 NAUSEA AND VOMITING 
 
Dose-dependent mild nausea and occasional vomiting have been reported.  Nausea was 
seen during the administration of hourly fractionated doses [21,30,31,34] or immediately 
after bolus administration [24,29,37] (i.e., in approximately 2/3 of patients given 60 
mg/kg and 1/3 of patients given 30 mg/kg).  Vomiting occurred about 12 hours later in a 
few cases and resolved by 24 hours.  The patients in this study will be evaluated and 
treated as indicated by the severity of their symptoms. 
 
4.7.3 LIVER FUNCTION TEST ABNORMALITIES 
 
Dose-dependent abnormalities in liver function tests have also been reported.  The most 
common abnormality was a rise in transaminases, usually AST [24,25,26,29,31,35,37].   
In two studies up to 2/3 of patients given 60 mg/kg and 1/5 of those given 30 mg/kg had 
elevated AST levels [21,30].  This rise was small in most other reports, but in these two 
studies (possibly describing the same series of patients), 3 patients had levels 3 to 5 times 
above normal.  In all studies but one, the abnormalities resolved 72 hours later:  5 of 18 
patients’ transaminases normalized in 10 days and one patient with a history of excessive 
alcohol intake had elevated levels at 30 days [34].  Only nine patients in these studies had 
transient elevations in bilirubin (less than two times normal).  No study correlated the 
liver function test abnormalities with the occurrence of nausea and vomiting in individual 
patients. Based on this experience, follow-up in our patients will consist of testing of liver 
function tests at 48 hours post tumor resection and, if > than Grade 3, weekly for 2 weeks  
and then at monthly intervals thereafter to resolution. 
4.7.4 OTHER EVENTS 
 
One patient developed a lichenoid reaction a week after oral ALA (60 mg/kg) which 
resolved after 3 weeks [34].  No abnormalities have been noted in serum electrolytes or 
hematologic indices. 
 
4.8 
ADVERSE EVENT REPORTING 
Patients will be monitored postoperatively for adverse events until 30 days after the study 
drug administration. Adverse events will be graded using the NCI CTC 3.0 criteria. In the 

 
CASE 1305 Protocol Version dated 09.15.17 Page 16 of 30 
 
case of any of the events described above, the patient will be examined by a study 
physician and appropriate medical action taken. The event will be recorded in the 
patient's study file with the date of occurrence, date of resolution, severity and the actions 
taken.  Any unexpected or serious adverse events, Grade 3 or above, or drug reaction 
beyond the scope of what was described above, will be reported on a MedWatch form to 
the Principal Investigator, the Case Comprehensive Cancer Center Clinical Trial 
Regulatory Affairs Office, Institutional Review Board, Case Comprehensive Cancer 
Center Data Safety and Toxicity Committee, Food and Drug Administration, DUSA 
Pharmaceuticals via Dr. Andrew Sloan. 
FDA Reporting 
The University Hospitals Cleveland Medical Center Principal Investigator, as holder of 
the IND, will be responsible for all communication with the FDA.  In accordance with 21 
CFR 312.32, the University Hospitals Cleveland Medical Center Principal Investigator is 
responsible for notifying the FDA of SAEs that are serious, unexpected (not listed in the 
Investigator Brochure or package insert) and judged to be related (i.e., possible, probable, 
definite) to the study drug.  Events meeting the following criteria need to be submitted to 
the FDA as Expedited IND Safety Reports.    
 
7 Calendar Day IND Safety Report 
Any unexpected fatal or life-threatening suspected adverse event represent especially 
important safety information and, therefore, must be reported more rapidly to FDA (21 
CFR 312.32(c)(2)).  Any unexpected fatal or life-threatening suspected adverse event 
must be reported to FDA no later than 7 calendar days after the University Hospitals 
Cleveland Medical Center] Principal Investigator initial receipt of the information (21 
CFR 312.32(c)(2)).  University Hospitals Cleveland Medical Center Principal 
Investigator will complete a Medwatch Form FDA 3500A and notify the FDA by 
telephone or facsimile transmission. 
 
15 Calendar Day IND Safety Report 
The timeframe for submitting an IND safety report to FDA and all participating 
investigators is no later than 15 calendar days after the University Hospitals Cleveland 
Medical Center Principal Investigator determines that the suspected adverse event or 
other information qualifies for reporting (21 CFR 312.32(c)(1)). This includes any 
serious, unexpected adverse events considered reasonably or possibly related to the 
investigational agent.  University Hospitals Cleveland Medical Center Principal 
Investigator will complete a Medwatch Form FDA 3500A and notify the FDA by 
telephone or facsimile transmission.  If FDA requests any additional data or information, 
the [University Hospitals Cleveland Medical Center Principal Investigator must submit it 
to FDA as soon as possible, but no later than 15 calendar days after receiving the request 
(21 CFR 312.32(c)(1)(v)).    
 
Follow-up IND Safety Report 
Any relevant additional information that the University Hospitals Cleveland Medical 
Center Principal Investigator obtains that pertains to a previously submitted IND safety 
report must 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)).  The University Hospitals 

 
CASE 1305 Protocol Version dated 09.15.17 Page 17 of 30 
 
Cleveland Medical Center Principal Investigator will maintain records of its efforts to 
obtain additional information. 
 
 
 
IND Annual Reports 
IND Annual Reports 
 
 
 
 
5.0  
MEASUREMENT OF EFFECT 
 
5.1 
ENDPOINTS 
 
Objectives: The endpoints are detection of residual tumor and of normal tissue. The 
primary endpoint will be correspondence between intensity of in vivo fluorescence and 
PpIX concentration and the pathologist’s quantification of tumor in biopsy specimens, 
e.g. percent tumor present. The measures used in the secondary objective will be PpIX 
concentration, g/mg and intra-operative fluorescence intensity.  Measures used in the 
third objective will be the biopsy specimen’s fluorescence intensity and the MR image 
enhancement at the biopsy site’s intraoperative stereotactic coordinates as determined by 
the surgeon. 
 
5.2 
DATA ANALYSIS 
 
5.2.1 FLUORESCENCE INTENSITY LEVELS VS. HISTOPATHOLOGY 
 
The data sets will be collated into the 4 study groups (2 ALA doses and 2 study arms--
i.e., newly diagnosed and recurrent diagnosis) for analysis purposes. 
 
Histopathology of the 1st part of the biopsies, performed by a single experienced 
neuropathologist (i.e., Dr. Mark Cohen) will be used as the gold standard. The white-
light, fluorescence images, and intra-operative PpIX concentration readings will be 
compared to assess the sensitivity and specificity for biopsy-confirmed visible tumor.  
 
5.2.2 Intra-operative vs. Ex Vivo PpIX EXTRACTION ASSAY 
 
The intra-operatively determined PpIX concentration will be compared to the intra-
operative imaging, ex vivo PpIX concentration, and MR enhancement images. This 
analysis will facilitate an assessment of the utility of lower ALA doses for enhancing 
subjective and/or quantitative contrast of tumor versus background fluorescence and/or 
PpIX concentration. 
 
Reporting Serious Problems to FDA 
Medwatch Form FDA 3500A: 
http://www.fda.gov/Safety/MedWatch/HowToReport/DownloadForms/default.htm 
Telephone: 1-800-332-1088 
Fax: 1-800-FDA-0178 
 
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 18 of 30 
 
 
5.2.3 CONFOCAL FLUORESCENCE MICROSCOPY VS. 
HISTOPATHOLOGY 
 
The confocal fluorescence microscopy images and adjacent H&E sections will be 
compared to determine the relationship between microscopic localization of PpIX 
fluorescence, concentration, and tumor cells.  
 
6.0 
STATISTICAL PARAMETERS 
 
6.1 
PATIENT DATA SET 
 
For each patient the following data will be captured for analysis  (collection timing in 
parens):  
1. Patient demographic information (at enrollment). 
2. Tumor/normal tissue classification for each biopsy by the neurosurgeon (intra-
operatively). 
3. Histology report on 1st part of biopsies (one to several weeks post-operatively). 
4. PpIX extraction assay 2nd part of biopsies (one to several months post-
operatively). 
5. Confocal microscopy fluorescence images on 3rd part of biopsies (one to several 
months post-operatively). 
6. White light images of biopsy sites (i.e. of the tissue area around the biopsy 
location; intra-operatively). 
7. Fluorescence images of biopsy sites over an area sufficient to clearly demarcate 
the region of interest (intra-operatively). 
8. PpIX concentration at biopsy site. 
9. Frameless stereotaxy coordinates of biopsies (intra-operatively). 
10. Magnetic Resonance appearance of biopsy at coordinates taken in #8 (one to 
several weeks post-operatively). 
11. Magnetic Resonance image volumes (i.e., pre-,  intra-, and post-operative; one to 
several weeks post-operatively). 
7.0  
DRUG FORMULATION AND PROCUREMENT 
 
7.1  
ALA FORMULATION AND TOXICITIES 
7.1.1 ALA Formula 
 
The chemical formula for ALA is: 
 
NH2CH2COCH2CH2COOH · HCl 
 
Three common synonyms also used to refer to ALA are: 
 
δ-Aminolevulinic acid Hydrochloride 
5-Amino-4-oxopentanoic acid Hydrochloride 

 
CASE 1305 Protocol Version dated 09.15.17 Page 19 of 30 
 
5-Aminolaevulinic acid Hydrochloride 
 
7.1.2  ALA PREPARATION 
 
ALA (5-amino levulinic acid) is provided in powder form. Patients receive ALA orally, 
dissolved in orange juice or sterile water (< 60 ml) [14,15,19-40]. ALA is stored in 
powder form by the investigational pharmacist. Before use the ALA is weighed by the 
pharmacist and the surgeon or his delegate. The ALA is then dissolved in orange juice or 
sterile water maximally 15 min prior to ingestion by the patient. 
 
7.2  
ALA SUPPLY 
 
ALA is provided by: 
 
DUSA Pharmaceuticals, Inc.  
25 Upton Drive  
Wilmington, MA 01887  
Telephone: (978) 657-7500 
 
7.3  
ALA STORAGE 
 
ALA is provided in powder form in 10 g vials. 
 
7.4  
ALA PROCRUREMENT 
 
Drug is supplied gratis by DUSA Pharmaceuticals, Inc. to our investigational pharmacy. 
8.0 
STATISTICAL CONSIDERATIONS 
 
8.1 
STATISTICAL ANALYSIS PLAN 
Primary objective: We will assess 2 doses of 5-ALA, 10mg/kg and 20mg/kg, to 
determine the optimum ALA dose in terms of both sensitivity and specificity for residual 
tumor.  The endpoint will be correspondence between intensity of in vivo fluorescence 
and PpIX concentration and the pathologist’s quantification of tumor in biopsy 
specimens, e.g. percent tumor present.  Receiver operating characteristic (ROC) curves 
will be calculated for each of the 2 combinations of ALA dose to estimate a level of 
fluorescence that distinguishes residual tumor from normal tissue with large true positive 
fraction (TPF) and small false positive fraction (FPF). A joint 95% confidence region for 
TPF FPF will be calculated, using α*=1-sqrt(1- α) = 0.975 and univariate confidence 
limits for the individual probabilities. Having chosen the cut-points for each of the 4 
combinations, positive and negative predictive values and diagnostic likelihood ratios 
will be calculated to describe the characteristics of each [18]. 
 
To compare the test characteristics of each ALA dose and to assess how the test 
characteristics are affected by demographic, disease, and surgical characteristics we will 

 
CASE 1305 Protocol Version dated 09.15.17 Page 20 of 30 
 
fit multivariable generalized mixed models to tumor and normal biopsy specimens using 
robust standard errors to account for the correlation of samples taken from the same 
individual. 
 
Secondary Objective: We will assess the correlation between the recorded in vivo 
qualitative assessment of flouresence signal from the neurosurgeon with the post-surgical 
(i.e., ex vivo) absolute PpIX concentration detected in ex vivo tissue biopsies.  The 
surgeon will subjectively score the intra-operatively observed fluorescence level as 
follows: 0, no fluorescence, 1, low fluorescence, 2, moderate fluorescence, or 3, high 
fluorescence [after 16].  We will first graph fluorescence against each PpIX concentration 
measurement (intraoperative and ex vivo) to evaluate the shape of the relationship. 
Additionally we will calculate a Spearman’s correlation coefficient with 95% confidence 
interval to assess the correlation between fluorescence against each PpIX concentration 
measurement (intraoperative and ex vivo).  The sensitivity and specificity of these 
observations will be determined [after 16], where the sensitivity of observable 
intraoperative PpIX fluorescence is the probability of observable intraoperative 
fluorescence given the presence of tumor cells in the tissue and specificity is the 
probability of no observable intraoperative fluorescence given that there are no tumor 
cells in the tissue [16]. We will also explore whether dose, patient, and disease 
characteristics affect the strength of the relationship between these measurements using 
multivariable generalized mixed models.  
  
Tertiary Objective: To determine the association  between the presence of fluorescence in 
the surgical cavity and the post-operative image enhancement on MRI. This includes the 
relationship between the amount and location of residual tumor detected by fluorescence, 
PpIX concentration, and intra-operative frameless stereotaxy following maximal 
resection versus the amount and location of tumor imaged post-operatively via CT and/or 
MRI.  Assessment of agreement between fluorescence, intra-operative PpIX 
concentration, and MR intensity will be explored using the radiologist’s assessment of 
MR intensity, measured on an ordinal scale. A multivariable ordinal logistic model will 
be fitted to assess the strength of the relationship and whether dose, patient or disease 
characteristics affect the relationship. 
 
All models for all objectives will be fitted with robust standard errors to account for 
correlation between specimens taken from the same individual. Statistical models will be 
examined for goodness of fit; residual and influence statistics will be computed to ensure 
that outlying values do not have a disproportionate effect on the final models. 
8.2 
SAMPLE SIZE AND STATISTICAL POWER   
Our study is powered appropriately for determination of the primary objective which 
involves the assessment of 2 doses of 5-ALA, 10mg/kg and 20mg/kg, to determine the 
optimum ALA dose in terms of both sensitivity and specificity for residual tumor.  We 
calculate the total sample size for 80% power at the 0.05 significance level, given that the 
sensitivity will decrease by 15% with decreasing ALA dose within patient group (i.e. 
newly diagnosed or recurrent).  Therefore our null hypothesis is that the sensitivity of the 
10mg/kg dose is less than or equal to 15% different from the 20 mg/kg dose and the 

 
CASE 1305 Protocol Version dated 09.15.17 Page 21 of 30 
 
alternative hypothesis is that the sensitivity of the 10mg/kg dose is greater than 15% 
different from the 20 mg/kg dose.  We also take into account having multiple biopsies per 
individual on study.  The total sample size per group per dose is as follows: 
 
The primary objective of this Phase II study is to estimate and compare the combined 
diagnostic accuracy of in-vivo qualitative assessment of fluorescent signal in detecting 
residual tumor when used by the neurosurgeon using  2 doses of 5-ALA:  10 mg/kg and 
20 mg/kg.   The in-vivo qualitative assessment by the neurosurgeon will be coded on an 
ordinal scale ranging from 0 (no signal) to 3 (highest signal), as was done in a similar 
study reported by Roberts et al (J Neurosurg 2010 Apr 9, epub ahead of print) using a 
dose of 20 mg/kg in newly diagnosed gliomas. As in Roberts et al, we assume a score of  
1 or higher will be considered positive.   In the study by Roberts et al (2010) the reported 
sensitivity and specificity were 0.75 and 0.71, respectively, using the histopathologic 
reading as gold standard.  In this study, because having high sensitivity and specificity 
are both important in terms of avoiding false negatives (which would involve not 
removing residual tissue) and false positives (removing normal tissue), the Youden index, 
defined as Sensitivity + Specificity -1 will be used as an overall  measure of diagnostic 
accuracy.  This study is designed so that if the dose levels of 10 and 20 mg/kg have true 
Youden indices that differ by 0.10 or more, then the probability is 80% or higher that the 
observed Youden index will be higher for the group with the higher actual index, i.e., the 
probability of selecting the correct dose level based on observed Youden indices will be 
80% or higher.    
For each patient, samples will be removed from six sites (2 center of tumor, 2 edge of 
tumor, and 2 adjacent normal tissue).  It is assumed that on average, three samples will be 
tumor positive based on pathology, and three will be tumor negative.  Thus, on average, 
each subject will contribute 3 true positive samples (used in estimating sensitivity), and 3 
true negatives (used in estimating specificity).  If n subjects per stratum (new or 
recurrent) are studied at a given dose, and π and θ are the sensitivity and specificity, we 
assume that the estimated sensitivity and specificity obtained as the proportions of true 
positives and true negatives are approximately normally distributed with means   π and θ 
and respective variances   π(1- π)(1+2)/3n, and θ (1- θ)(1+2)/3n, where  is the within-
subject correlation of binary responses obtained for tissues on the same subject. This 
formula can be derived for example assuming a beta-binomial model to model extra-
binomial variability. Here the term (1+2) is the design effect where the coefficient of  
is m-1, where m is the number of true positive or true negative samples per subject.  
Given specified values of sensitivity and specificity for the two doses reflecting a 
scenario where Youden indices differ between doses, the probability that the better dose 
has the higher observed Youden index can be determined, and the sample size n 
determined such that this probability is high. 
 
Table 1 below presents required numbers of patients per group in order to have 80% 
probability of correctly identifying the optimal dose, when the Youden indices of the two 
doses differ by 0.10, using a range of plausible values for sensitivity and specificity, for 
intrasubject correlations of  =0.1 and 0.2.  Based on these results it can be seen that 
with sample sizes of 30 glioblastoma multiformes (GBMs) there is a 80% or higher 

 
CASE 1305 Protocol Version dated 09.15.17 Page 22 of 30 
 
chance of correctly identifying the optimal dose when Youden indices differ by 0.10 
or more. Assuming sample size n=30 and intrasubject correlation =0.2, the 95% 
confidence intervals for π=0.75, θ=0.70, and youden index=0.45 are (0.64, 0.86), 
(0.59, 0.81), and (0.23, 0.67), respectively.  
 
Table 1.  Required sample size per group to correctly identify the optimal dose with 
probability of 80%, when Youden indices differ by 0.10 between doses, for various 
combinations of sensitivity and specificity and intrac-subject correlation, . 
 
Dose=20 mg/kg 
Dose=10 mg/kg 
 
ΔYouden 
(20 – 10) 
Required # subjects 
per group 
Sens 
Spec 
Youden 
Sens 
Spec 
Youden 
=0.10 
=0.20 
0.75 
0.70 
0.45 
0.60 
0.75 
0.35 
0.1 
23 
27 
0.75 
0.70 
0.45 
0.65 
0.70 
0.35 
0.1 
24 
28 
0.70 
0.70 
0.40 
0.60 
0.70 
0.30 
0.1 
24 
28 
0.70 
0.70 
0.40 
0.55 
0.75 
0.30 
0.1 
24 
28 
0.65 
0.70 
0.35 
0.55 
0.70 
0.25 
0.1 
25 
30 
0.65 
0.70 
0.35 
0.50 
0.75 
0.25 
0.1 
25 
29 
0.75 
0.70 
0.45 
0.70 
0.85 
0.55 
-0.1 
21 
24 
0.75 
0.70 
0.45 
0.75 
0.80 
0.55 
-0.1 
21 
25 
0.70 
0.70 
0.40 
0.70 
0.80 
0.50 
-0.1 
22 
26 
0.70 
0.70 
0.40 
0.65 
0.85 
0.50 
-0.1 
22 
26 
0.65 
0.75 
0.40 
0.60 
0.90 
0.50 
-0.1 
21 
25 
0.65 
0.70 
0.35 
0.60 
0.85 
0.45 
-0.1 
22 
26 
 
However, based on imaging, it is difficult to distinguish Grade IV malignant gliomas 
(GBMs) from  Grade III tumors, otherwise known as anaplastic astrocytomas (AAs) in 
newly diagnosed patients.   Traditionally, AA’s account for 10-15% of all malignant 
gliomas with a rate of 11.1% reported for the US in 2010 (41).   Assuming that 15% of all 
patients with newly diagnosed malignant gliomas have AA rather than GBM, we will 
need to accrue 35 patients to accrue 30 patients with newly diagnosed GBM.    In the case 
of patients with recurrent malignant gliomas, all patients presenting with histological 
evidence of glioma who cary a diagnosis of GBM from an earlier surgery are considered 
to have “recurrent GBM” by convention.  Patients carrying a diagnosis of AA who have 
recurrent have degenerated to GBM approximately half the time.   Accordingly, of 33 
patients with recurrent malignant glioma, 29 would be expected to have recurrent GBM, 
while 5 would be expected to have recurrent tumor arising from AA.    Of the 5 with AA, 
two or three would be expected to have progressed to GBM.    
 
Thus, in order to accrue 30 patients with newly diagnosed GBM, we will need to accrue 
35 patients with newly diagnosed malignant glioma.   In order to accrue 30 patients with 
recurrent GBM, we will need to accrue 33 patients with recurrent malignant glioma. 
 
There are no planned interim analyses for efficacy because this is not a therapeutic trial. 
Monitoring of safety and trial progress will be the responsibility of the Data and Safety 
Toxicity Committee. 
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 23 of 30 
 
9.0  
PLAN FOR INCLUSION OF VULNERABLE POPULATIONS 
 
9.1  
Plan for Inclusion of Illiterate Individuals: 
 
We may potentially enroll illiterate individuals to this trial.  During the consent process, 
the consent form will be read to the patient by the study coordinator or consenting 
physician.  This consent process will be witnessed by a third party with no interest in this 
trial who will sign the consent form as a witness.   
 
 
9.2  
Plan for Inclusion of Non-English Speaking Individuals: 
 
We may potentially enroll non-English speaking individuals to this trial.  If a short form 
consent document is available in a particular patient’s language we will use the short 
form consent document to consent the patient.  This short form will only be used once.   
When using the short form consent document, a translator will be present during the 
discussion (or UHCMC language line will be used).  We will not use family members as 
translators for medical research.  The translator will verbally review the full length 
English informed consent form with the subject and will discuss all parameters of 
participation.  If the subject agrees to participate, the study staff (person obtaining 
consent), the translator, and the PI will sign the English consent.  The subject will sign 
the short form only.  The documents will be kept together in the subject’s research file. 
 
For any subsequent non-English speaking patients, we will translate the consent form and 
obtain IRB approval prior to the consent process.  Once the translated consent form has 
been reviewed and approved by the IRB, we will only then consent the patient with the 
consent form written in the patient’s native language.   
 
9.3  
Plan for Inclusion of Pregnant Women and Fetuses: 
 
Pregnant women are excluded from participation in this trial.  See exclusion criteria in 
Section 3.2.  If a woman becomes pregnant while participating in this trial, all study 
treatment will be discontinued.  We will continue to follow the patient as outlined in the 
protocol. Research personnel will not offer the subject any type of inducement (monetary 
or otherwise) to terminate the pregnancy nor will the research personnel have any part in 
any decisions regarding timing, method, procedures used to terminate the pregnancy, or 
any part in determining viability of a neonate. 
 
10.0 
RECORDS TO BE KEPT 
 
Patients will be identified by site and study number. Demographic information 
corresponding to study numbers will be kept in a locked office in the research area of the 
clinical trial site. However, health records may be given to and inspected by the Case 
Western Reserve University Institutional Review Board for purposes of monitoring the 
study. All records will be kept for 3 years after the completion of the trial. 
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 24 of 30 
 
All intraoperative procedures will be carried at University Hospitals of Cleveland 
(Cleveland, OH).  The tissue biopsies required for histopathology will be given to Dr. Dr. 
Mark Cohen. Tissue biopsies for spectrofluorimetry and confocal fluorescence 
microscopy will be transported to Dr. Wilson’s laboratory at the Ontario Cancer 
Institute/Princess Margaret Hospital.  No identifying information will be transferred; the 
biopsies will be labeled with the patient study number and the image number. These 
biopsies are not required for patient care purposes. The tissues will be handled according 
to institutional safety protocols. These are the same procedures that have been used for 
our investigator group’s preceding glioma Photofrin-PDT trial. 
 
The study will commence only after approval by the University Hospitals of Cleveland 
IRB.  
 
10.1 
Electronic Medical Records 
 
This study will access electronic medical records systems to obtain medical information 
for the subjects enrolled to this study.  In order to insure patient safety, investigators and 
study personnel must have up-to-the-minute health information for subjects enrolled to 
this study.  Therefore, electronic medical records must be utilized to obtain medical 
information in a timely manner.  The following electronic systems will be used:  IDX 
program to access scheduling information; UH Physician Portal to access lab results and 
physician notes; UHHS Information Network (mainframe) to access lab results and 
physician notes; PACS to access radiological imagining results; and MySecureCare 
(Sunrise Clinical Manager) to access some or all of the above information when this 
application is fully functional.  This information will be obtained by the PI, co-
investigators, study coordinator, and/or data manager for this study via password-
protected login.  Access to these systems is required for the life of this research study.  
Information obtained from electronic systems will be copied (scheduling information) 
into the Ireland Cancer Center Clinical Trials Unit research chart and/or printed (lab 
results, physician notes, etc.) and stored in the research chart.  Research charts are kept 
secure and destroyed according to UH policy.  All study personnel involved in this 
research will adhere to the UH policies regarding confidentiality and Protected Health 
Information.  Some study personnel may access the above electronic systems for research 
purposes only. 
11.0 
LITERATURE CITED 
1.  Shapiro, W.R., et al., Randomized trial of three chemotherapy regimens and two 
radiotherapy regimens and two radiotherapy regimens in postoperative treatment of 
malignant glioma. Brain Tumor Cooperative Group Trial 8001. J Neurosurg, 1989. 
71(1): p. 1-9. 
2. Walker, M.D., et al., Randomized comparisons of radiotherapy and nitrosoureas for 
the treatment of malignant glioma after surgery. N Engl J Med, 1980. 303(23): p. 
1323-9. 

 
CASE 1305 Protocol Version dated 09.15.17 Page 25 of 30 
 
3. Nitta, T. and K. Sato, Prognostic implications of the extent of surgical resection in 
patients with intracranial malignant gliomas. Cancer, 1995. 75(11): p. 2727-31. 
4. Devaux, B.C., J.R. O'Fallon, and P.J. Kelly, Resection, biopsy, and survival in 
malignant glial neoplasms. A retrospective study of clinical parameters, therapy, and 
outcome. Journal of Neurosurgery, 1993. 78(5): p. 767-75. 
5. Rostomily, R.C., et al., Multimodality management of recurrent adult malignant 
gliomas: results of a phase II multiagent chemotherapy study and analysis of 
cytoreductive surgery. Neurosurgery, 1994. 35(3): p. 378-88; discussion 388. 
6. Yoshida, J., et al., Long-term follow-up results of 175 patients with malignant 
glioma: importance of radical tumour resection and postoperative adjuvant therapy 
with interferon, ACNU and radiation. Acta Neurochir, 1994. 127(1-2): p. 55-9. 
7. Lacroix, M., et al., A multivariate analysis of 416 patients with glioblastoma 
multiforme: prognosis, extend of resection and survival. J. Neurosurg., 2001. 
95(August): p. 190190-198. 
8. Stummer, W., et al., Technical principles for protoporphyrin-IX-fluorescence guided 
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41. http://www.cbtrus.org/2009EighteenStates/Table5.pdf 
 
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 28 of 30 
 
APPENDICES 
 
 
 
12.0 
APPENDIX I: SKIN PHOTOSENSITIVITY DUE TO ALA 
 
Patient Information and Instructions 
 
The drug you receive during this study is called ALA (which stands for Aminolevulinic Acid).  It 
collects in all tissues, including the skin.  The amount of ALA that will be used is much less than 
the usual amount given when ALA is used for treatments. However, after receiving the ALA, we 
recommend the following precautions for 24 hours: 
 
 
1.  No direct sun exposure. 
 
During this time, exposure to sunlight may cause a severe sunburn.  This can be prevented by 
protection from sunlight, either by using thick clothing or by not going outside.  Sun protection 
creams of any strength are not effective. Sunglasses, facemasks, gloves, long sleeves and pants 
are required during this time if you are out in the sun. Direct sunlight through a window should 
also be avoided. Brief times (one minute or less) in weak sunlight are safe and may help 
desensitize you to sunlight.  
 
2.  No exposure to strong indoor lights. 
 
During this time, exposure to strong indoor lighting may also cause a severe burn.  Examples of 
such lighting are: tanning booths, a dentist’s lamp, a doctor’s examining lamp, an operating room 
lamp, a direct spotlight or floodlight, and strong bathroom vanity lights.  Ordinary room lights 
and reading lamps with shades are safe and will also help to desensitize your skin.  You do not 
need to stay in a completely dark room or to cover your skin and eyes while indoors. In the 
operating room, appropriate precautions will be taken to avoid strong light exposure.  
 
 
At the end of the 24-hour period, we recommend that you expose a small area of skin (like the 
back of your hand) to sunlight for five minutes.  If there is no sunburn by the next day, you 
probably have no more ALA left in your skin. You may then return to normal light exposure. 
 
If at any time a prickly or burning feeling or a skin reddening is experienced after exposure to the 
bright light, please call us at the numbers below and remain in dim light until symptoms have 
disappeared.  
 
If you experience any other unusual reaction please call the numbers below. 
 
Contact numbers: 
 
Dr. Andrew Sloan 
 
 
 
 
  
phone no: (216) 844-6054 
Christopher Murphy, RN 
 
 
  
 
             phone no: 216-983-
3021 
resident on call (for night-time and weekend emergencies)  
phone no: (216) 844-1000, 
ask for pager number: 30153 
 

 
CASE 1305 Protocol Version dated 09.15.17 Page 29 of 30 
 
 
APPENDIX II: DESCRIPTION OF THE FLUORESCENCE IMAGING SYSTEM 
 
 
This fluorescence imaging system is 
specifically designed for surgical 
procedures. It consists of a compact 
surgical camera that has 720x480 
pixels resolution and displays 30 
frames/sec. It is multispectral. i.e. it 
can 
simultaneously 
detect 
3 
wavelength bands simultaneously 
(400-500nm, 500-600nm, and 600-
700nm). This system is hand-held 
and a standard laparoscope is used 
as entrance optics. The excitation 
light source is a 300W xenon lamp, 
similar 
to 
lamps 
used 
for 
endoscopy, which has a spinning 
filter wheel so that is 2 different 
wavelength bands are used for 
excitation (440nm,  80nm FWHM 
(150mW) 
and 
470nm, 
60nm 
FWHM 
(240mW)). 
When 
the 
illuminated area is 6 cm2 the 
maximum irradiance is 40mW.cm-2. 
Quantitation of the fluorescence 
image is achieved by ratiometric image processing algorithms 
on the different images obtained by multi-excitation and multi 
detection. Additionally, the images can be quantified by use of 
a sterile fluorescence standard (i.e. a fluorescent crystal 
attached to a fiber optic cable) with known fluorescence that 
will be placed in the surgical cavity to serve as a reference.  
The computer and software allow to display and store the 
fluorescence images as well as more quantitative ratio images in real time. In addition, 
these images can be displayed on the monitors available in the OR for surgical guidance.      
 
The system is has electrical approvals by Canadian Standards Association (CSA) and 
Underwriter Laboratories Inc. (UL) and is currently being used in a clinical trial for 
fluorescence guided detection of residual tumor after prostatectomy using ALA-PpIX at 
University Health Network, Toronto (Dr. J. Trachtenberg and Dr. B.C. Wilson).   
 
This system will be used in this clinical trial. 

 
CASE 1305 Protocol Version dated 09.15.17 Page 30 of 30 
 
APPENDIX III: STUDY FLOW CHART  
This flow chart details the fluorescence imaging/biopsy procedures. 
(WLI: white light imaging; FLI: fluorescence imaging) 
 
 
 
 
            13. Save fluorescence video and repeat steps 7 
 
 
8. Identify biopsy site using FLI  
9. Place FL standard pointing to biopsy site 
10. Record coordinates of biopsy site with 
11. Record Fluorescence video with taking biopsy 
12. Split biopsy in 3 parts, fix and label.  
13. Repeat above steps so that 6 biopsies are taken 
1.Identify biopsy site using FLI 
2. Place FL standard pointing to biopsy site 
3. Record coordinates of biopsy site with stereotaxy 
4. Record fluorescence video while taking biopsy 
5. Split biopsy in 3 parts, fix and label  
6. Repeat above steps so that 6 biopsies are taken 
Preoperative-CT/MRI scan, 
ALA administration 
~ 50% tumor resection 
Time point A: Bulk tumor measurements 
 
Operation End 
Post-operative CT/MRI scan 
 
Maximum tumor resection 
Time point B: Tumor bed measurements 
7. Continue tumor resection