A Randomized Double-Blind Controlled Trial of Everolimus in Individuals With PTEN Mutations (RAD001XUS257T)
Phase: PHASE1
Conditions: PTEN Gene Mutation, PTEN Hamartoma Tumor Syndrome
Sponsor: Boston Children's Hospital

RDCRN Protocol #7904        
             Everolimus with PTEN Mutations 
          Version Date: 05Feb20 
 
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Rare Diseases Clinical Research Network (RDCRN) 
 
 
 
A Randomized Double-Blind Controlled Trial of 
Everolimus in Individuals with PTEN Mutations 
(EverolimusXUS257T) 
 
Developmental Synaptopathies Consortium  
 
 
 
Antonio Hardan, MD 
401 Quarry Road 
Palo Alto, CA 94305 
650-724-8919 
 
 
 
 
 
 
 
 
 
 
 
 
This protocol is for research purposes only, and should not be copied, 
redistributed or used for any other purpose.  The procedures in this 
protocol are intended only for use by Consortium investigators in 
carefully controlled settings.  The Chair of this study should be 
consulted before using or attempting any procedure in this protocol. 

RDCRN Protocol #7904        
             Everolimus with PTEN Mutations 
          Version Date: 05Feb20 
 
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Table of Contents 
 
Participating Institutions Contact Information .......................................................................................................... 6 
Protocol Synopsis .................................................................................................................................................... 6 
1. Introduction ........................................................................................................................................................ 10 
2. Objectives and Endpoints.................................................................................................................................. 11 
2.2 Secondary objective .................................................................................................................................... 11 
2.3 Exploratory objective ................................................................................................................................... 11 
3. Background ....................................................................................................................................................... 12 
3.1 Overview of Germline Heterozygous PTEN Gene Mutations ..................................................................... 12 
3.2 Preclinical Studies of Neurocognition in PTEN Gene Mutations ................................................................ 13 
3.3 Clinical Studies of Neurocognition in PTEN Gene Mutations ..................................................................... 13 
3.4 Overview of Everolimus .............................................................................................................................. 13 
3.4.1 Everolimus ........................................................................................................................................... 13 
3.4.2 The role of mTOR pathway in tumorigenesis ...................................................................................... 14 
3.4.3 Preclinical Studies of Everolimus ........................................................................................................ 15 
3.4.4 Brain Penetration ................................................................................................................................. 15 
3.4.5 Everolimus Pharmacokinetics ............................................................................................................. 16 
3.4.6 Pharmacodynamic Studies .................................................................................................................. 17 
3.4.7 Phase I investigation of Safety, Tolerability, and Molecular Pharmacodynamic effects in patients with 
advanced solid tumors .................................................................................................................................. 17 
3.4.8 A randomized, double-blind, placebo-controlled study of RAD001 in the treatment of patients with 
subependymal giant cell astrocytes (SEGA) associated with Tuberous Sclerosis Complex (TSC) ............ 17 
3.4.9 Phase I Study of Everolimus in Pediatric Patients with Refractory Solid Tumors ............................... 18 
3.5 Study Rationale ........................................................................................................................................... 18 
4 Design and Methods .......................................................................................................................................... 19 
4.1 Study Rationale ........................................................................................................................................... 19 
4.2 Pre-Treatment Phase (Screening) .............................................................................................................. 19 
4.3 Blinded Treatment Phase ........................................................................................................................... 20 
4.4 Open Label Phase ...................................................................................................................................... 21 
4.5 Follow-Up Phase ......................................................................................................................................... 21 
4.6 Patient Selection and Inclusion/ Exclusion Criteria..................................................................................... 21 
4.6.1 Double-Blind Inclusion Criteria ............................................................................................................ 22 
4.6.2 Double-Blind Exclusion Criteria ........................................................................................................... 22 
4.6.3 Open-Label Phase Inclusion Criteria ................................................................................................... 24 
4.6.4 Screening for Hepatitis B, Hepatitis C, and HIV .................................................................................. 24 
5. Recruitment Methods ........................................................................................................................................ 25 
5.1 Retention Strategies ................................................................................................................................... 26 
6. Description of Study Treatments or Exposures/Predictors ............................................................................... 26 
6.1 Everolimus Administration .......................................................................................................................... 26 
6.2 Treatment Course Information .................................................................................................................... 26 
6.3 Known Undesirable Effects of Study Drug/Treatment ................................................................................ 28 

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6.3.1 Adverse Drug Reactions (ADRs) Reported for Previous Tuberous Sclerosis Complex Studies ........ 28 
6.3.2 Management of Infections ................................................................................................................... 29 
6.3.3 Management of Skin Toxicity .............................................................................................................. 30 
6.3.4 Management of Hypersensitivity Reactions ........................................................................................ 30 
6.3.5 Angioedema with Concomitant Use of Angiotensin-Converting Enzyme (ACE) Inhibitors ................. 30 
6.3.6 Renal Failure Events ........................................................................................................................... 30 
6.3.7 Management of Mouth Ulcers/Stomatitis/Oral Mucositis .................................................................... 30 
6.3.8 Management of Hyperlipidemia and Hyperglycemia ........................................................................... 31 
6.3.9 Management of Diarrhea ..................................................................................................................... 31 
6.3.10 Dosing Modifications In Case of Treatment Related Toxicities ......................................................... 31 
6.3.11 Management of Non-Infectious Pneumonitis .................................................................................... 32 
6.3.12 Follow-Up for Toxicities ..................................................................................................................... 33 
7. Description of Study Procedures ....................................................................................................................... 33 
7.1 Written Informed Consent ........................................................................................................................... 33 
7.2 Process of Consent ..................................................................................................................................... 34 
7.3 Remote Consent Process ........................................................................................................................... 34 
7.4 Patient Numbering ...................................................................................................................................... 34 
7.5 Randomization Procedure .......................................................................................................................... 35 
7.6 Double-Blind Treatment Assignment Unblinding ........................................................................................ 35 
7.6.1 Emergency Unblinding Treatment Assignment ....................................................................................... 35 
7.7 Treating the Patient ..................................................................................................................................... 35 
7.7.1 Study Drug Administration ................................................................................................................... 35 
7.7.2 Treatment Compliance ........................................................................................................................ 36 
7.7.3 Other Permitted Study Drug Adjustments ........................................................................................... 36 
7.7.4 Concomitant Medications .................................................................................................................... 37 
7.7.5 Concomitant Medication Dose Adjustments ........................................................................................ 38 
7.7.6 Study Drug Interruption or Discontinuation ......................................................................................... 39 
7.7.7 Withdrawal from the study and study evaluation completion .............................................................. 39 
7.7.8 Study Discontinuation .......................................................................................................................... 39 
7.8 Good Clinical Practice and Human Subjects Training ................................................................................ 40 
7.9 Neuropsychological Evaluations ................................................................................................................. 40 
7.9.1 Training and Reliability of Independent Evaluations (IEs) ................................................................... 40 
7.9.2 Research Report.................................................................................................................................. 40 
7.9.3 Main Efficacy Outcomes ...................................................................................................................... 40 
7.9.4 Additional Secondary Efficacy Outcomes ........................................................................................... 41 
7.10 Dermoscopy Assessment ......................................................................................................................... 43 
7.11 Eye Tracking (Optional) ............................................................................................................................ 43 
7.12 Resting State Encephalography and Auditory Evoked Potential (Optional) ............................................. 44 
7.13 Safety Outcomes ...................................................................................................................................... 45 
7.14 Vital Signs ................................................................................................................................................. 46 

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7.15 Body Surface Area .................................................................................................................................... 46 
7.16 Laboratory Evaluations ............................................................................................................................. 46 
7.16.1 Hepatitis and HIV Screening ............................................................................................................. 47 
7.16.2 Hematology........................................................................................................................................ 47 
7.16.3 Coagulation........................................................................................................................................ 47 
7.16.5 Biochemistry and Lipid Profile ........................................................................................................... 48 
7.16.7 Urinalysis ........................................................................................................................................... 48 
7.16.8 Blood Sample for Everolimus (Everolimus Levels) ........................................................................... 48 
7.16.9 Additional Blood Sample for PTEN Associated Proteins .................................................................. 49 
7.16.10 Additional Blood Sample for Insulin Like Growth Factor Binding Protein 2 (IGFBP-2) ................... 49 
7.16.11 Microbiome/Mycobiome Sample Collection .................................................................................... 49 
7.17 Data from Samples ................................................................................................................................... 50 
7.18 Medical Records ....................................................................................................................................... 50 
7.19 Follow-Up Questionnaires ......................................................................................................................... 50 
7.20 Data Collection .......................................................................................................................................... 55 
7.21 Inclusion/Exclusion Criteria ....................................................................................................................... 55 
7.22 Patient Demographics/Clinical Interview (Medical History) ...................................................................... 55 
7.23 GCP Statement ......................................................................................................................................... 56 
8. Benefits.............................................................................................................................................................. 56 
9. Risks .................................................................................................................................................................. 56 
10. Study Records Retention ................................................................................................................................ 56 
11. Sample Destruction ......................................................................................................................................... 56 
12. Protocol Deviations ......................................................................................................................................... 56 
13. Adverse Event Criteria and Reporting Procedures ......................................................................................... 56 
13.1 Definitions and Reporting .......................................................................................................................... 57 
13.2 Laboratory Test Abnormalities .................................................................................................................. 57 
13.3 Serious Adverse Event Criteria and Reporting Procedures ..................................................................... 58 
13.3.1 Definitions .......................................................................................................................................... 58 
13.3.2 Adverse Event Reporting ................................................................................................................... 58 
13.4 RDCRN Adverse Event Data Management System (AEDAMS) .............................................................. 60 
13.5 Unanticipated Problem Reporting ............................................................................................................. 60 
13.6 Pregnancies .............................................................................................................................................. 60 
14. Study Oversight ............................................................................................................................................... 61 
15. Data and Safety Monitoring Committee (DSMC) ............................................................................................ 61 
16. Data Management and Coordinating Center and Database ........................................................................... 61 
16.1 Registration ............................................................................................................................................... 61 
16.2 Data Elements .......................................................................................................................................... 62 
16.3 Data Entry ................................................................................................................................................. 62 
16.4 Laboratory Data Flow ................................................................................................................................ 62 
16.5 Data Quality and Monitoring Measures .................................................................................................... 62 

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16.6 Quality Control Method ............................................................................................................................. 63 
17. Data Analysis Plan, Statistical Power and Sample Considerations ................................................................ 63 
17.1 Power Estimation ...................................................................................................................................... 65 
18. Protocol Amendments or Changes in Study Conduct ..................................................................................... 66 
19. Compensation ................................................................................................................................................. 66 
20. Privacy Provisions ........................................................................................................................................... 66 
21. Confidentiality Provisions ................................................................................................................................ 67 
21.1 Certificate of Confidentiality ...................................................................................................................... 67 
References ............................................................................................................................................................ 69 
 
 
 

RDCRN Protocol #7904        
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Participating Institutions Contact Information 
 
 
Principal Investigator: Antonio Hardan, M.D. 
Co-Investigator: Lawrence Fung, M.D.  
Contact:  Jaelyn Edwards |  Robin Libove  |  Lakshmi Vrittamani 
Institution: 
Stanford University 
Address: 
401 Quarry Road 
Palo Alto, CA 94305 
Phone: 
650-736-1235 
Email: 
jedwrds@stanford.edu | rlibove@stanford.edu | vrittama@stanford.edu  
 
 
Principal Investigator: Rabi Hanna, M.D. 
Co-Investigator: Charis Eng, M.D., Ph.D. 
Contact: 
Komeisha Rose 
Institution: 
Cleveland Clinic 
Address: 
9500 Euclid Ave., S20  
Cleveland, OH 44195   
Phone: 
216-445-8798 
Email: 
rosek2@ccf.org 
 
 
Principal Investigator: Mustafa Sahin, M.D., Ph.D. 
Co-Investigator: Siddharth Srivastava, M.D.  
Contact: 
Gregory Geisel 
Institution: 
Boston Children’s Hospital 
Address: 
3 Blackfan Circle 
CLS14064 
Boston, MA 02115 
Phone: 
617-919-1476 
Email: 
gregory.geisel@childrens.harvard.edu    
 
 
Unblinded Study Physician: Sarah Spence, M.D. 
Back-up Unblinded Physicians: Kiran Maski, M.D.   |   David Franz, M.D. 
Contact: 
Grace Bazin 
Institution: 
Boston Children’s Hospital 
Address: 
3 Blackfan Circle 
CLS14064 
Boston, MA 02115 
Phone: 
617-355-5230 
Email: 
Grace.Bazin@childrens.harvard.edu  
 
 
 
The RDCRN Data Management and Coordinating Center (DMCC) designated by the 
National Institutes of Health 
 
 
Protocol Synopsis 
Interventional Synopsis 
Protocol Number: 
7904 
Protocol Title: 
A Randomized Double-Blind Controlled Trial of Everolimus in 
Individuals with PTEN Mutations (EVEROLIMUSXUS257T) 
Study Chair: 
Antonio Hardan, M.D.  
Statistician: 
Booil Jo, Ph.D. 
Consortium: 
Developmental Synaptopathies Consortium 

RDCRN Protocol #7904        
             Everolimus with PTEN Mutations 
          Version Date: 05Feb20 
 
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Participating Sites: 
Boston Children's Hospital  
Cleveland Clinic 
Stanford University Medical Center 
Activation Date: 
June 9, 2017 
Current Status: 
Open to Enrollment  
Sample Size: 
40 randomized 
Target Enrollment Period: 
2 Years 
Study Design: 
Interventional, Phase I/II, Multi-Center 
Primary Study Objective: 
To evaluate the safety of everolimus compared with placebo in 
patients with PTEN mutations 
Secondary Study 
Objective(s): 
To evaluate the efficacy of everolimus on neurocognition and 
behavior in patients with PTEN mutations compared to placebo as 
measured by standardized, direct and indirect neurocognitive tools 
and behavioral measures (processing speed/working memory). 
Study Population and Main 
Eligibility/ Exclusion 
Criteria: 
Double-Blind Inclusion Criteria 
 
1. Male and female outpatients between 5 and 45 years of age 
(inclusive); 
2. Pathogenic PTEN mutation confirmed by clinical genetic 
testing; 
3. Participant must be able to complete one of the following three 
standardized assessments: Conners’ Continuous Performance 
Tasks (CPT-3-mean reaction time), Stanford Binet (SB-5; 
working memory), or the Purdue Pegboard Test;  
4. Performance below the age-adjusted population mean on at 
least one of the above standardized measure: attention (CPT-
3, mean reaction time), working memory (SB5), or fine motor 
skills (Purdue Pegboard Test; either dominant hand, non-
dominant hand, or both hands); 
5. Adequate bone marrow function as shown by:  
a. platelets ≥ 80,000/mm3 
b. absolute neutrophil count ≥ 1,000/mm3  
c. hemoglobin ≥ 9 g/dL 
6. Adequate liver function as shown by: 
a. Total serum bilirubin < 1.5 x ULN  
b. AST and ALT levels < 2.5 x ULN 
c. 
INR ≤ 2  
7. Adequate renal function: serum creatinine < 1.5 x ULN,  
8. Signed informed consent obtained prior to any screening 
procedures; 
9. Individuals on psychotropic and anti-epileptic medications 
should maintain a stable dose for at least 2 months prior to the 
screening visit; 
10. Negative serum pregnancy test for females at screening and 
no plans to become pregnant or conceive a child while 
participating in the study. The effects of mTOR inhibitors on the 
developing fetus at the doses used in this study are unknown. 
For this reason, women of child-bearing potential and men 
must agree to use adequate contraception prior to study entry 
and for the duration of the study. Estrogen-containing oral 
contraceptives are not recommended in women enrolled in this 
study.  Abstinence or two  effective non-estrogen or barrier 
methods of contraception (such as condoms + spermicidal 
foam) must be used; 
11. No anticipated changes in the frequency and intensity of 
existing interventions such as behavioral and developmental 
treatments, in home services, and speech therapy;  

RDCRN Protocol #7904        
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12. No planned changes in school placement;  
13. For individuals under 18 or who are otherwise incapable, there 
must be an available caregiver who can reliably bring subject 
to clinic visits and provide trustworthy data 
14. Able to communicate fluently in English  
 
Double-Blind Exclusion Criteria 
15. Patients currently receiving anticancer therapies or who have 
received anticancer therapies within 4 weeks of the start of 
Everolimus 
(including 
chemotherapy, 
radiation 
therapy, 
antibody based therapy, etc.); 
16. Known intolerance or hypersensitivity to Everolimus or other 
rapamycin analogs (e.g. sirolimus, temsirolimus); 
17. Known impairment of gastrointestinal (GI) function or GI 
disease that may significantly alter the absorption of oral 
Everolimus; 
18. Uncontrolled diabetes mellitus as defined by HbA1c >8% 
despite adequate therapy. Patients with a known history of 
impaired fasting glucose or diabetes mellitus (DM) may be 
included, however blood glucose and antidiabetic treatment 
must be monitored closely throughout the trial and adjusted as 
necessary; 
19. Patient with uncontrolled hyperlipidemia: fasting serum 
cholesterol > 300 mg/dL OR >7.75 mmol/L AND fasting 
triglycerides > 2.5 x ULN.  
20. Patients who have any severe and/or uncontrolled medical or 
psychiatric conditions (see section 4.6 for additional details) 
21. Chronic 
treatment 
with 
corticosteroids 
or 
other 
immunosuppressive agents. Topical or inhaled corticosteroids 
are allowed; 
22. Known history of or seropositivity for Hepatitis B, Hepatitis C, 
or HIV; 
23. Patients who have received live attenuated vaccines within 1 
week of start of Everolimus and during the study. Patient 
should also avoid close contact with others who have received 
live attenuated vaccines. Examples of live attenuated vaccines 
include intranasal influenza, measles, mumps, rubella, oral 
polio, BCG, yellow fever, varicella and TY21a typhoid vaccines; 
24. Patients who have a history of another primary malignancy, 
with the exceptions of:  
a. non-melanoma skin cancer,  
b. and carcinoma in situ of the cervix, uteri, or breast from 
which the patient has been disease free for ≥3 years; 
25. Planned changes to concomitant medications;   
26. Prior or concomitant therapy with known or possible anti-
mTOR activity, including rapamycin (sirolimus);  
27. Concomitant therapy with strong inhibitor (e.g., cyclosporine 
and ketoconazole) or inducer of CYP3A; 
28. Active infection at time of enrollment;  
29. Patients with a history of non-compliance to medical regimens 
or who are considered potentially unreliable or will not be able 
to complete the entire study; 
30. Patients who are currently part of or have participated in any 
clinical investigation with an investigational drug within 1 month 
prior to dosing; 

RDCRN Protocol #7904        
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31. Pregnant or nursing (lactating) women; 
32. Women of child-bearing potential (WOCBP), defined as all 
women physiologically capable of becoming pregnant, must 
use highly effective methods of contraception during the study 
and 8 weeks after. Highly effective contraception methods 
include: 
a. A combination of any two of the following: 
33. Use of oral, injected or implanted hormonal non-estrogen 
containing methods of contraception or; 
34. Placement of an intrauterine device (IUD) or intrauterine 
system (IUS); 
35. Barrier methods of contraception: condom or occlusive cap 
(diaphragm 
or 
cervical/vault 
caps) 
with 
spermicidal 
foam/gel/film/cream/ vaginal suppository; 
36. Total abstinence or; 
37. Male/female sterilization. 
 
Women are considered post-menopausal and not 
of child-bearing potential if they have had 12 
months of natural (spontaneous) amenorrhea 
with an appropriate clinical profile (e.g. age 
appropriate, history of vasomotor symptoms) or 
have had surgical bilateral oophorectomy (with or 
without hysterectomy) or tubal ligation at least six 
weeks prior to randomization. In the case of 
oophorectomy alone, only when the reproductive 
status of the woman has been confirmed by 
follow up hormone level assessment is she 
considered not of child-bearing potential. 
38. Male patients whose sexual partner(s) are WOCBP who are 
not willing to use adequate contraception, during the study and 
for 8 weeks after the end of treatment 
39. Major surgery, radiation therapy, or stereotactic radio-surgery 
within previous 4 weeks at time of screening 
40. Neurosurgery within prior 6 months at time of screening. 
 
Open-Label Inclusion Criteria 
41. Patients who completed Double-Blind phase of the study and 
were assigned to the placebo treatment arm; 
42. Verbal consent (and assent, as appropriate) obtained prior to 
any open-label phase study procedures. 
 
Treatment  
Agent 
Everolimus 
Dosage, schedule, route of 
administration- 
4.5mg/m2/day, to the nearest 2.5mg, orally  
Safety Issues- 
The safety profile is characterized by manageable adverse events 
(AEs). These AEs are generally reversible and non-cumulative.  
Adverse events most frequently observed with everolimus are 
stomatitis, 
mouth 
ulcers, 
acne, 
infection, 
and 
menstrual 
irregularities. Adverse events that are also common include: rash, 
diarrhea, fatigue, asthenia, nausea, peripheral edema, decreased 
appetite, headache, dysgeusia, epistaxis, mucosal inflammation, 
pneumonitis, weight decreased, vomiting, pruritus, cough, dyspnea, 
dry skin, nail disorder, aggression, and pyrexia.  
Overall, the most frequently observed laboratory abnormalities 
include: decreased hematology parameters including hemoglobin, 

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lymphocytes, 
platelets, 
and 
neutrophils; 
increased 
clinical 
chemistry parameters including cholesterol, triglycerides, glucose, 
aspartate transaminases, creatinine, alanine transaminases, and 
bilirubin; and decreased clinical chemistry parameters including 
phosphate and potassium.  
The majority of these AEs have been of mild to moderate severity 
(NCI CTC grade 1-2). Recommendations for dose adjustments, 
should any of these treatment related adverse events occur, are 
given in Table 3. 
Primary Outcome 
Measures: 
The primary endpoint will be study drop-out rate due to side effects, 
comparing everolimus vs. placebo. We will also determine the 
frequency of adverse events by type and severity. We predict that 
the rate of adverse events will be no more than 10% higher in the 
everolimus group compared to placebo but overall severity will be 
minimal. 
Secondary Outcome 
Measures: 
The main efficacy endpoint will be a neurocognitive composite. 
This composite will be computed in two ways: 1) an average for 
working memory (SB-5 working memory), processing speed (CPT 
mean reaction time) and fine motor (Purdue Pegboard-average of 
both hands) subtests, and 2) the same average as above excepted  
weighted 2/3 and an additional average based on all other 
available 
neurocognitive 
testing 
measures 
(receptive 
and 
expressive language, non-verbal ability, verbal learning, sustained 
attention, impulsivity, and visuomotor skills, etc.) weighted 1/3. 
Additional efficacy endpoints will examine the effect of everolimus 
on overall global clinical improvement, autism symptoms, other 
behavior problems, and adaptive abilities as measured by 
validated, standardized instruments including the Clinical Global 
Impressions-Improvement scale, Autism Diagnostic Observation 
Schedule – Second Edition (ADOS-2 calibrated severity score), 
Social Responsiveness Scale-Second Edition (SRS-2), Adult/Child 
Behavior Checklist (ABCL/CBCL), Wide Range Assessment of 
Memory and Learning-2 (WRAM-L2) and Vineland Adaptive 
Behavior Scales, 3rd Edition (VABS-III). 
Statistical Considerations 
(sample size and analysis 
plan): 
See data analysis section of protocol 
Sponsors (federal, state, 
foundation and industry 
support): 
National Institutes of Health; Novartis Pharmaceuticals; PTEN 
Research Foundation  
 
1. Introduction 
 
This is a Phase I/II 6-month, randomized, double-blind placebo-controlled trial of everolimus in patients, ages 5 
to 45 years (inclusive) with a PTEN mutation, with safety and neurocognition as the primary endpoints.  
 
Germline heterozygous phosphatase and tensin homolog (PTEN) gene mutations are associated with a 
spectrum of clinical disorders characterized by neurocognitive deficits, autism symptomatology, skin lesions, 
macrocephaly, hamartomatous overgrowth of tissues, and an increased risk of cancers. The neurocognitive and 
neurobehavioral deficits are associated with very high morbidity, health care cost, and impact on the quality of 
life of individuals with PTEN and their families. Currently, there are no approved agents for the treatment of 
these neurocognitive and neurodevelopmental deficits in PTEN. 
 
This protocol addresses a key area of interest: Pharmacological treatments of a well-defined subgroup with 
autism spectrum disorder. It will leverage an existing investigation that aims at examining the natural history 
study of individuals with autism and germline heterozygous PTEN mutations (BCH IRB-P00013150: Natural 
History Study of Individuals with Autism and Germline Heterozygous PTEN Mutations). The purpose of this 

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phase II pilot study will be to establish short-term safety of everolimus treatment in individuals with germline 
PTEN mutations and evaluate associated cognitive and behavioral changes in this population to generate 
plausible hypotheses to be tested in a future Phase III confirmatory trial. This protocol also mirrors a previous 
clinical trial utilizing everolimus in the tuberous sclerosis population (BCH 10-06-0247 Randomized Double-Blind 
Phase 2 Trial of RAD001 for Neurocognition in Individuals with Tuberous Sclerosis Complex).        
       
In this investigation, we propose a 6-month randomized, double-blind, multi-site study to evaluate the safety and 
efficacy of a rapamycin analogue, everolimus, to treat neurocognitive and social deficits in a total of 60 patients 
(age range 5-45 years) with a PTEN mutation with a maximum of 40 participants randomized to treatment. By 
maintaining a lower age limit of 5 years, we can ensure a more accurate assessment of cognitive and behavioral 
outcomes, streamline the neurobehavioral assessment battery, and with minimal loss to outcome measure 
scope and sensitivity, similar to the strategy we employed in a previous everolimus trial in tuberous-sclerosis 
complex (TSC) (NCT01289912), performed by PI Mustafa Sahin, M.D., Ph.D.   
 
This investigation involves 3 participating sites (Stanford University, site PI: Antonio Hardan, M.D.; Cleveland 
Clinic, site PI: Rabi Hanna, M.D.; and Boston Children’s Hospital, site PI: Mustafa Sahin, M.D., Ph.D.). 
 
2. Objectives and Endpoints  
 
2.1 Primary objective 
 
To evaluate the safety of everolimus compared with placebo in patients with PTEN mutations focusing on NCI 
CTCAE Grade 3 and 4 adverse events, serious adverse events, and Grade 3 and 4 laboratory toxicities. The 
primary endpoint will be study drop-out rate due to side effects, comparing everolimus vs. placebo. Based on 
the published literature and our experience in prior TSC trials we hypothesize that the drop-out rate due to side 
effects in those receiving everolimus will be similar to those in the placebo group with minimal effect size (< 10% 
difference). We will also determine the frequency of adverse events by type and severity. We predict that the 
rate of adverse events will be no more than 10% higher in the everolimus group compared to placebo, but 
overall severity will be minimal. 
 
2.2 Secondary objective 
 
To evaluate the efficacy of everolimus on neurocognition and behavior in individuals with PTEN mutations 
compared to placebo as measured by standardized, direct and indirect neurocognitive tools and behavioral 
measures. The main efficacy endpoint will be a composite index score of neurocognitive function that will be 
computed in two ways. The first will be an average of measures evaluating working memory (Stanford-Binet 
Intelligence Scales, Fifth Edition; SB-5 working memory subscale), processing speed (Conners’ Continuous 
Performance Test, Third Edition; CPT-3 mean reaction time), and fine motor skills (Purdue Pegboard Test 
average of both hands). The second will include the above average weighted by two-thirds and an average of 
the remaining standardized, norm-referenced neurocognitive measures (e.g., non-verbal ability, visuomotor 
skills, verbal learning, receptive and expressive language) weighted by one-third.  We hypothesize that 
individuals receiving everolimus will show more improvement, relative to those taking placebo, in the composite 
index.   
 
Additional efficacy endpoints will examine the effect of everolimus on overall global clinical improvement, autism 
symptoms, other behavior problems, and adaptive abilities as measured by validated, standardized instruments 
including the Clinical Global Impressions-Improvement scale, Autism Diagnostic Observation Schedule – 
Second Edition (ADOS-2 calibrated severity score), Social Responsiveness Scale - Second Edition (SRS-2), 
Adult/Child Behavior Checklist (CBCL ACF/CBCL), Wide Range Assessment of Memory and Learning, Second 
Edition (WRAML-2), and Vineland Adaptive Behavior Scales, Third Edition (VABS-III). We will also assess the 
effect of everolimus treatment on eye tracking tasks, resting state EEG, and task related EEG through auditory 
evoked potentials (AEP). 
 
2.3 Exploratory objective 
 
To determine if PTEN-associated pathway molecules (PI3K/AKT, mTOR, MAPK), RNA levels, protein levels, 
and differences in the gut microbiome and mycobiome are affected by everolimus treatment and correlate with 
clinical improvements. For PTEN biochemistry, the primary endpoint will be relative change in total and 

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phosphorylated protein levels following treatment when compared to baseline in isolated peripheral blood 
mononuclear cells (PBMCs), which will then be correlated with everolimus levels. Other endpoints will be 
derived from each method and correlative studies will assess whether adverse events or neurocognitive 
changes are associated with a particular RNA, protein, microbiome or mycobiome profile or everolimus levels. 
The comprehensive analysis of these objectives will depend on the availability of additional funds, and at this 
time, samples will be collected and stored as per protocol.    
 
3. Background  
 
3.1 Overview of Germline Heterozygous PTEN Gene Mutations 
 
Phosphatase and tensin homolog (PTEN) gene germline mutations are associated with a spectrum of clinical 
disorders characterized by neurocognitive deficits, intellectual disability, autism symptomatology, skin lesions, 
macrocephaly, hamartomatous overgrowth of tissues, and an increased risk of cancers.1-11 In humans, PTEN-
related research has historically focused on physical manifestations of the disease and the pathophysiology and 
treatment of the hamartomatous lesions that arise in affected patients and their predisposition for malignancy. In 
contrast, there is much less research to date focused on the behavioral and cognitive features, and their 
treatment.2,4,8 Currently no effective therapies for individuals with germline PTEN mutations exist to target 
neurocognitive and social deficits, and effective interventions that target the core biologic alterations are crucial 
to optimize the short and long-term outcome of individuals affected with mutations in this gene. 
 
PTEN is a dual specificity phosphatase that inhibits both the PI3K/AKT/mTOR and MAPK pathways, which, in 
turn, inhibit neural cell growth, proliferation, and synaptic function.1,2  Historically, research efforts have focused 
on examining the physical manifestations resulting from these mutations, but there has been little interest in the 
behavioral and cognitive features and their treatment. In fact, the cancer risk is managed by high risk 
surveillance and early detection or prophylactic surgery and there is currently no effective treatment for the 
severe neurocognitive and social/communication deficits observed in PTEN mutation.3,4   
 
Pervasive deficits in children with PTEN mutation include impairments in social development, cognitive 
functioning, and communication.5 Active research is ongoing to identify disease-specific pharmacological 
treatment and medications, such as everolimus, are promising to target the core deficits observed in this 
disorder. Everolimus is an mTOR inhibitor that has been examined in different medical conditions including 
tuberous sclerosis. Because it may address underlying physiological abnormalities, everolimus may also 
improve core features of individuals with PTEN mutations and inform future research on pathophysiology of this 
disorder. 
 
In patients with PTEN mutation, the mTOR pathway is abnormally upregulated and interferes with normal cell 
growth, proliferation, and function.  Much is known regarding upstream and downstream regulation of mTOR.  
There are two structurally and functionally distinct multi-protein signaling complexes, mTORC1 (mTOR complex 
1, rapamycin sensitive)31 and mTORC2 (mTOR complex 2, rapamycin insensitive). mTORC1 is mainly 
activated via the PI3 kinase pathway through AKT (also known as PKB, protein kinase B) and the tuberous 
sclerosis complex (TSC1/TSC2). Activated AKT phosphorylates TSC2, which leads to the dissociation of 
TSC1/TSC2 complex, thus inhibiting the ability of TSC2 to act as a GTPase activating protein. This allows Rheb, 
a small G-protein, to remain in a GTP bound state and to activate mTORC1. AKT can also activate mTORC1 by 
PRAS40 phosphorylation, thereby relieving the PRAS40-mediated inhibition of mTORC1.32,33 mTORC2 
(mTOR complex 2) is activated through a currently unknown mechanism, possibly by receptor tyrosine kinase 
(RTK) signaling. It has been suggested that mTORC2 phosphorylates and activates a different pool of AKT that 
is not upstream of mTORC1. PHLPP phosphatase plays the role of a negative regulator. mTORC2 is rapamycin 
insensitive and is required for the organization of the actin cytoskeleton.31  Like TSC, PTEN deficiency is an 
attractive target for treatment with mTOR inhibitors due the PTEN’s role through AKT to regulate specifically 
mTORC1, which is sensitive to rapamycin and analogs such as everolimus.   
 
PTEN regulates a varied set of intracellular processes.2,16-19  It is hypothesized that when dysfunctional, PTEN 
protein interacts with the protein of a second gene known as Tp53 to dampen energy production in neurons. 
This severe stress leads to a spike in harmful mitochondrial DNA changes and abnormal levels of energy 
production in the cerebellum and hippocampus, brain regions critical for social behavior and cognition. In 
addition, PTEN interaction with p53 triggers deficiencies in additional proteins linked to learning disabilities, 
including autism. Multiple lines of evidence support its role in cognitive development and in social behavior.5,8,9,13  

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While PTEN-associated pathways and their impact of cell growth and proliferation have been well described in 
the context of cancer,6-8 much less attention has been given to understanding the role of PTEN in 
neurocognition,5,9,10 It is not yet known which pathway components are most disrupted in human PTEN to cause 
neuronal synaptic dysfunction8,11-13 and the associated autism spectrum disorder (ASD) and intellectual disability 
(ID) common with PTEN loss-of-function.14,15   
 
3.2 Preclinical Studies of Neurocognition in PTEN Gene Mutations 
 
Multiple murine CNS-conditional knock-out models have established the role of PTEN in learning and control of 
social behavior.12,13 Using a germline model, female PTEN heterozygous animals exhibited decreased social 
behavior.20 More recently, a germline model which results in inappropriate PTEN subcellular localization showed 
inappropriate social behavior and clumsiness, reminiscent of a subset of PTEN mutation positive ASD 
patients.21-24  Additionally, PTEN loss in mature neurons have led to diminished social behavior, an effect 
replicated in a model of PTEN loss in neuronal precursors.25,26_ENREF_5 The Nse-cre x PtenloxP/loxP model also 
shows decreased recognition learning and increased anxiety.27 Interestingly, inhibition of mTOR complex 1, a 
downstream target of AKT signaling, improved social behavior in this model.26  
 
3.3 Clinical Studies of Neurocognition in PTEN Gene Mutations  
 
In humans, PTEN-related research has historically focused on physical manifestations of the disease and the 
pathophysiology and treatment of the hamartomatous lesions that arise in affected patients and their 
predisposition for malignancy. In contrast, there is much less research to date focused on the behavioral and 
cognitive features, and their treatment.5,9,28 Currently no effective therapies for individuals with germline PTEN 
mutations exist to target neurocognitive and behavioral deficits, and effective interventions that target the core 
biologic alterations are crucial to optimize the short and long-term outcome of individuals affected with mutations 
in this gene. 
 
3.4 Overview of Everolimus  
 
3.4.1 Everolimus 
 
Everolimus is a novel derivative of rapamycin. Everolimus has been in clinical development since 1996 as an 
immunosuppressant in solid organ transplantation. In 2003, everolimus was approved in Europe (trade name: 
Certican) via the Mutual Recognition Procedure (MRP) for the prevention of organ rejection in patients with 
renal and cardiac transplantation. Certican is also approved in Australia, South Africa, the Middle East, Central 
and South America, the Caribbean and some Asian countries. 
 
Everolimus 2.5mg, 5mg, 7.5mg, and 10mg tablets were approved under the trade name Afinitor® for patients 
with advanced renal cell carcinoma (RCC) after failure of treatment with Sutent® (sunitinib) or Nexavar® 
(sorafenib) in the US, EU, and several other countries and is undergoing registration in other regions worldwide.    
 
In 2010, Afinitor® received United States (US) approval for patients with subependymal giant cell astrocytoma 
(SEGA) associated with tuberous sclerosis complex (TSC). Everolimus is also available as Votubia® in the 
European Union (EU) for patients with SEGA associated with TSC who require therapeutic intervention but are 
not candidates for curative surgical resection. Afinitor® was approved for “progressive pancreatic 
neuroendocrine tumor (PNET) in patients with unresectable, locally advanced, or metastatic disease” in 2011 in 
various countries, including the US and Europe. In 2012 Afinitor® received approval for the treatment of 
postmenopausal women with advanced hormone receptor-positive, HER2- negative breast cancer (advanced 
HR+ BC) in combination with exemestane, after failure of treatment with letrozole or anastrozole. Furthermore, 
in 2012, Afinitor® received approval for the treatment of adult patients with TSC who have renal 
angiomyolipoma not requiring immediate surgery. AFINITOR® DISEPERZ was approved in 2018 (April 10th) for 
adjunctive treatment of adult and pediatric patients aged 2 year and older with TSC-associated partial-onset 
seizures. 
 
Everolimus is also approved to treat hamartomatous lesions in tuberous sclerosis complex (TSC), a genetic 
disorder with synaptic disruption and cognitive and behavioral features similar to those of PTEN.  At the cellular 
and molecular level, everolimus acts as a signal transduction inhibitor. Everolimus binds to FKB12 to selectively 

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inhibit mTOR, a key and a highly conservative serine-threonine kinase. Everolimus selectively inhibits mTOR 
(mammalian target of rapamycin), specifically targeting the mTOR-raptor signal transduction complex. mTOR is 
a key serine-threonine kinase in the PI3K/AKT signaling cascade, which is known to be dysregulated in a wide 
spectrum of human cancers.10mTOR is present in all cells and is a central regulator of protein synthesis and 
ultimately cell growth, cell proliferation, angiogenesis and cell survival. mTOR is currently the only known target 
of everolimus.29   
 
3.4.2 The role of mTOR pathway in tumorigenesis   
 
The target of everolimus is mTOR (mammalian target of rapamycin), a serine-threonine kinase implicated in the 
PI3K/AKT pathway known to be active in numerous neoplastic conditions. Driven largely by growth factors 
acting upstream, the PI3K/AKT/mTOR pathway modifies downstream signaling events involved in the regulation 
of cell-cycling, cell growth and cell survival mechanisms. 
 
An important aspect of the antitumor effect of everolimus is its potential to act on both tumor cells directly (to 
inhibit growth) and indirectly (by inhibiting angiogenesis).  The observation of in vivo sensitivity of xenografts 
comprised of cells demonstrating resistance to everolimus in vitro is attributed to the drug’s potential to act on 
the vascular component of the supporting peritumoral stroma.  The anti-angiogenic property of everolimus has 
been confirmed through experiments demonstrating the effect of everolimus in countering VEGF-induced 
proliferation of human umbilical vein endothelial cells (HUVECs) in vitro, VEGF-driven angiogenesis in a 
chamber implant murine model and neovascularization in a murine orthotopic melanoma model. 
 
At the cellular and molecular level, everolimus acts as a signal transduction inhibitor. The target of everolimus is 
mTOR (mammalian target of rapamycin), a serine-threonine kinase which is a member of the larger PI3K 
(phosphatidylinositol 3-kinase) family and present in all cells. Everolimus selectively inhibits mTOR, which 
regulates cell growth, proliferation and survival. The mTOR kinase is mainly activated via the 
phosphatidylinositol 3-kinase (PI3K) pathway through AKT/PKB and the tuberous sclerosis complex (TSC1/2). 
Mutations in these components or in PTEN, a negative regulator of PI3 kinase, may result in their dysregulation. 
Abnormal functioning of various components of the signaling pathways contributes to the pathophysiology of 
numerous human cancers. Various preclinical models have confirmed the role of this pathway in tumor 
development. 
 
The main known functions of mTOR include:30 
 
mTOR functions as a sensor of mitogens, growth factors, energy and nutrient levels, facilitating cell-cycle 
progression from G1 - S phase in appropriate growth conditions. 
 
The PI3K (mTOR) pathway itself is frequently deregulated in many human cancers, and oncogenic 
transformation may sensitize tumor cells to mTOR inhibitors. 
 
The mTOR pathway is involved in the production of pro-angiogenic factors (e.g. VEGF) and in endothelial 
cell growth and proliferation. 
 
Through inactivating eukaryotic initiation factor 4E binding proteins and activating the 40S ribosomal S6 
kinases (e.g. p70S6K1), mTOR regulates protein translation. 
 
The regulation of mTOR signaling is complex and involves positive regulators, such as AKT, that phosphorylate 
and inactivate negative regulators such as the Tuberous Sclerosis Complex (TSC1/TSC2). 
 
The PI3K/AKT/mTOR pathway is known to be dysregulated in numerous proliferative disorders including 
cancer. Molecular epidemiological studies have also shown that activation of the PI3K/AKT/mTOR pathway is 
frequently associated with worsening prognosis through resistance to treatment, disease extension and disease 
progression. A variety of preclinical models have confirmed the role of this pathway in tumor development. It has 
also been demonstrated that constitutional activation of kinases such as AKT can lead to inexorable 
development of cancers resembling those which in patients are characterized by frequent activation of the same 
kinases. This is complemented by the demonstration of the antitumor activity of kinase inhibitors acting on the 
pathway in vitro and in vivo preclinical models.  
 
Table 1 Drug substance 
Chemical name: 
(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-
dihydroxy-12-(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-

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methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-
hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24, 
26,28-tetraene-2,3,10,14,20-pentaone 
International non-proprietary 
name 
Everolimus 
 
3.4.3 Preclinical Studies of Everolimus  
  
Everolimus inhibits the proliferation of a range of human tumor cell lines in vitro including lines originating from 
lung, breast, prostate, colon, melanoma and glioblastoma.  Everolimus also inhibits the proliferation of human 
umbilical vein endothelial cells (HUVECs) in vitro, with particular potency against VEGF-induced proliferation 
suggesting that everolimus may also act as an anti-angiogenic agent. The anti-angiogenic activity of everolimus 
was confirmed in vivo. Everolimus selectively inhibited VEGF-dependent angiogenic response at well-tolerated 
doses. Mice with primary and metastatic tumors treated with everolimus showed a significant reduction in blood 
vessel density when compared to controls. 
 
Everolimus administered daily by mouth was a potent inhibitor of tumor growth, at well tolerated doses, in 11 
different mouse xenograft models (including pancreatic, colon, epidermoid, lung and melanoma) and two 
syngeneic models (rat pancreatic, mouse orthotopic melanoma).  These models included tumor lines considered 
sensitive and “relatively resistant” in vitro. In general, everolimus was better tolerated in mouse xenograft 
models than standard cytotoxic agents (i.e., doxorubicin and 5-fluorouracil), while possessing similar anti-tumor 
activity.  Additionally, activity in a VEGF-impregnated subcutaneous implant model of angiogenesis and reduced 
vascularity (vessel density) of everolimus-treated tumors (murine melanoma) provided evidence of in vivo 
effects of angiogenesis. 
 
All significant adverse events observed in toxicology studies with everolimus in mice, rats, monkeys and mini-
pigs were consistent with its anticipated pharmacological action as an anti-proliferative and immunosuppressant, 
and were at least in part reversible after a 2- or 4-week recovery period with the exception of the changes in 
male reproductive organs, most notably testes. 
 
In the published article, Pharmacological Inhibition of mTORC1 Suppresses Anatomical, Cellular, and 
Behavioral Abnormalities in Neural-Specific Pten Knock-Out Mice, Zhou et al. made a PTEN KO mouse that has 
neurological deficits. They treated the mice either at a presymptomatic stage (5–6 weeks old) or a symptomatic 
stage (adult 10–12 weeks old). They showed that rapamycin can rescue macrocephaly even in the adult group. 
Furthermore, rapamycin was able to significantly reverse neuronal hypertrophy in older mice, resulting in 
restoration of much of the hippocampal structure. 
 
Another article published by Nguyen and Anderson, mTOR-dependent alterations of Kv1.1 subunit expression in 
the neuronal subset-specific Pten knockout mouse model of cortical dysplasia with epilepsy, showed that PTEN 
loss alters Potassium channel expression in the in the hippocampus. They demonstrate that mTOR inhibition 
with rapamycin treatment at early (4-5 weeks) and late (9-10 weeks) stages of the pathology normalized Kv1.1 
protein levels in NS-Pten KO mice to WT levels 
 
 
3.4.4 Brain Penetration 
 
Everolimus penetration into brain tissue has been evaluated in mice and rats. Non-tumor bearing BALB/c mice 
were administered everolimus once at 5 mg/kg orally or 1 mg/kg intravenously, and blood and tissues were 
obtained at times after drug administration. The extent of penetration (AUCbrain/AUCblood) was 1.8% and 5.2% 
after oral and intravenous administration.  Despite the relatively low penetration, everolimus brain tissue 
concentrations (69 ng/g after oral; 8 ng/g after intravenous) are within range of the IC50 values for PTEN-/- 
glioblastomas for at least 9 hours and above the IC50 values for PTEN+/+ mice for at least 2 hours.34,35 
 
In Wistar rats, the brain everolimus distribution was related to drug dose and time after intravenous drug 
administration. At everolimus dosages up to 1 mg/kg, the blood and brain tissue concentrations increased 
linearly; thereafter, a nonlinear increase was noted in both.  The higher everolimus brain tissue concentrations 
at higher intravenous dosages are consistent with a saturation of an efflux pump present in brain capillary 
endothelial cells. The kinetics of everolimus brain uptake were established by measuring blood and brain tissue 

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concentrations of 3H everolimus at various times after an intravenous bolus dosage. Everolimus was rapidly 
distributed in the brain with slow efflux. At 168 hours after the dose, significant brain levels of 6 ng/g were 
detected, without any significant blood levels. Although up to 24 hours no metabolites are noted in the brain, at 
168 hours they represent 60% of total drug in the brain.34  
 
Although everolimus penetration into the brain is low, after a 1.0 mg intravenous dose of everolimus in Wistar 
rats, brain tissue concentrations exceeded the in vitro antiproliferative IC50 for HUVEC cells and a panel of 
PTEN-/- glioblastoma cell lines for 168 hours, and selected PTEN+/+ cell lines for 24 hours.35,36 
 
 
 
3.4.5 Everolimus Pharmacokinetics 
 
The pharmacokinetic characteristics of everolimus have been extensively investigated in the context of the 
drug’s development as an immunosuppressant in solid organ transplantation where everolimus was 
administered twice daily as a part of an immunosuppressant multi-drug regimen consistently including 
cyclosporin A and glucocorticoids. Recent Phase I studies provide steady-state pharmacokinetics for both the 
weekly and daily schedules at varying dose levels in patients with advanced cancers. 
 
Everolimus is rapidly absorbed after oral administration, with a median time to peak blood levels (tmax) of 1-2 
hours post dose. The extent of absorption is estimated to be above 11%.  The area under the blood 
concentration-time curve (AUC) is dose-proportional over the dose range tested while maximum blood 
concentration C2h appears to plateau at dose levels higher than 20 mg. The terminal half-life in cancer patients 
averaged 30 hours, which is similar to that in healthy subjects. Inter-patient variability is moderate with the 
coefficient of variation (CV) of approximately 50%.  A high-fat meal altered the absorption of everolimus with 
1.3-hour delay in tmax, a 60% reduction in C2h and a 16% reduction in AUC. In whole blood, approximately 80% 
of everolimus is contained in red blood cells. Of the fraction of drug contained in plasma, 74% is protein-bound. 
The apparent distribution volume (Vz/F) after a single dose was 4.7 L/kg. Everolimus is eliminated by 
metabolism, mainly by hydroxylation, then excreted into the feces >80%. 
 
Everolimus is mainly metabolized by CYP3A4 in the liver and to some extent in the intestinal wall. Everolimus is 
also a substrate of P-glycoprotein (P-gp). Therefore, absorption and subsequent elimination of systematically 
absorbed everolimus may be influenced by medicinal products that interact with CYP3A4 and/or P-glycoprotein. 
In vitro studies showed that everolimus is a competitive inhibitor of CYP3A4 and of CYP2D6 substrates, 
potentially increasing the concentrations of medicinal products eliminated by these enzymes. In two phase III 
clinical trials in patients following kidney transplantation, strong inhibitors of CYP3A4 (azoles, antifungals, 
cyclosporine, erythromycin) have been shown to reduce the clearance of everolimus therapy thereby increasing 
everolimus blood levels. Similarly, Rifampin, a strong inducer of CYP3A4, increases the clearance of everolimus 
thereby reducing everolimus blood levels. Caution should be exercised when co-administering everolimus with 
CYP3A4 inhibitors or inducers. 
 
Patients with PTEN may be treated with P450 enzyme-inducing antiepileptic drugs (EIAEDs) which lead to an 
increase of everolimus apparent clearance. Commonly used EIAEDs which are used in this population include:  
1. Phenytoin (Dylantin®, Dilantin Kapseals®, Dilantin® Infatabs®, Eptoin®, Epanutin®, Diphenin®, 
Dipheninum®, Phenytek®),  
2. Mephenytoin (Mesantoin®),  
3. Carbamazepine (Tegretol®, Biston®, Calepsin®, Carbatrol®, Epitol®, Equetro®, Finlepsin®, Sirtal®, 
Stazepine®, Telesmin®, Teril®, Timonil®, Trimonil®, Epimaz®, and Degranol®),  
4. Phenobarbital (Luminal®),  
5. Pentobarbital (Nembutal®),  
6. Primidone (Mysoline®),  
7. Oxycarbazepine (Trileptal®).  
 
Patients receiving EIAEDs show decreased plasma levels of several medications when administered at 
conventional doses. This may, in turn, lead to ineffective dosing.  
 
Everolimus pharmacokinetics in transplant patients were investigated in special populations such as subjects 
with hepatic or renal impairment, various ethnic groups and pediatric renal transplant patients. In subjects with 

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mild or moderate hepatic impairment, mean AUC to everolimus is increased 2-fold whilst renal impairment does 
not affect the pharmacokinetics of everolimus. Age, weight (both over the adult range) and gender do not affect 
the pharmacokinetics of everolimus to a clinically relevant extent. Also, pharmacokinetics does not alter in Asian 
patients whereas black patients have 21% higher clearance compared to non-blacks. A single, escalating-dose 
study in Japanese subjects did not show a significant difference in dose normalized systemic exposure. 
 
The pharmacokinetic parameters derived for everolimus given daily are summarized in Table 2. 
 
Table 2   Steady-state everolimus pharmacokinetics (daily dosing) 
Parameter 
5 mg 
10 mg 
N 
4 
6 
tmax (h) 
1 (1) 
1 (1-6) 
Cmin
ss (ng/mL) 
5.4 ± 1.8 
13.2 ± 7.9 
Cmax
ss (ng/mL) 
32 ± 9 
61 ± 17 
Cmax
ss/Dose (ng/mL/mg) 
6.4 ± 1.8 
6.1 ± 1.7 
AUCss (ng·h/mL) 
238 ± 77 
514 ± 231 
AUCss/Dose (ng·h/mL/mg) 
48 ± 15 
51 ± 23 
Cavg
ss (ng/mL) 
9.9 ± 3.2 
21.4 ± 9.6 
Values are median (range) for tmax and mean ± standard deviation for all others. 
Dose-normalized parameters are per mg.  is 24 h 
 
3.4.6 Pharmacodynamic Studies 
 
Pharmacokinetic/pharmacodynamic modeling based on inhibition in a peripheral biomarker (S6 kinase inhibition 
in peripheral blood mononuclear cells) suggests that 10 mg daily should be an adequate dose to produce a 
high-degree of sustained target inhibition. Furthermore, molecular pharmacodynamic (MPD) studies using 
immunohistochemistry (IHC) in biopsied tumor tissue assessed the degree of inhibition and its duration (for p-
S6, p-4E-BP1 and p-AKT expression) with the daily and weekly dosing. The pathologist was blinded for the 
biopsy sequence. There was almost complete inhibition of p-S6 at all doses and schedules studied (p=0.001). 
Preliminary results suggest a dose-related decrease in p-4E-BP1 and increase in p-AKT expression with 
maximal effect at 10 mg daily and ≥ 50 mg weekly.  
 
3.4.7 Phase I investigation of Safety, Tolerability, and Molecular Pharmacodynamic effects in patients with 
advanced solid tumors 
 
In this study, protocol CRAD001C2107, molecular changes were subsequently investigated through serial 
biopsying of tumors before and while on treatment.37 Biopsying of tumors on treatment took place at week 4 of 
treatment (pharmacokinetic steady-state). All patients underwent a 24-hr post-dose biopsy. Patients following 
the weekly regimen had a further biopsy on Day 4-5 during the same week. Molecular activity was measured by 
immunohistochemistry. In the absence of a reliable technique for measuring mTOR phosphorylation itself, the 
phosphorylation status of downstream markers S6 and eIF4G, for which reliable antibodies exist, was selected 
as reflecting the immediate pharmacodynamic effect of everolimus. Also measured were changes in the 
phosphorylation status of upstream AKT and the proliferation index Ki67. The daily regimen was associated with 
a high inhibition of p-S6 and p-eIF4G at 5mg/d and a complete inhibition at 10mg/d. In patients on the weekly 
schedule, p-S6 inhibition was complete and sustained at all dose levels while that of p-eIF4G was complete and 
sustained at 50mg/d but not at 20mg/wk. On both regimens numerous patients demonstrated apparent up-
regulation of AKT which tended, however, not to persevere in the patients at 50mg/wk. The proliferation index 
was reduced in most patients, recovering in some of those on the 50mg/wk regimen. 
 
3.4.8 A randomized, double-blind, placebo-controlled study of RAD001 in the treatment of patients with 
subependymal giant cell astrocytes (SEGA) associated with Tuberous Sclerosis Complex (TSC) 
 
In the phase I Study CRAD001C2413, (IND #70,714) everolimus was administered orally at a daily dose of 2.1, 
3, 5 and 6.5 mg/m2 in cohorts of three to six patients per dosage level (Fouladi et al., 2007). The MTD for this 
population was 5 mg/m2. At the starting dose of 3 mg/m2 readily reversible grade 3 and 4 dose-limiting toxicities 
(DLTs) at 3 mg/m2 included reversible hypokalemia (n = 1, grade 4) and hypophosphatemia (n = 2, grade 3) 
were observed in platinum pretreated patients, resulting in dose de-escalation to 2.1 mg/m2. The definition of 
DLT was amended to exclude grade 3 or 4 electrolyte abnormalities that resolved to grade 2 or below within 7 

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days of interrupting treatment, allowing further dosage escalation. Dose de-escalation to 2.1 mg/m2 led to no 
further DLTs in three assessable patients. Three more assessable patients were then enrolled at 3 mg/m2 and 5 
mg/m2, each with no DLTs. At 6.5 mg/m2, the DLTs included grade 3 events of elevation of ALT (n = 1), 
mucositis (n = 1), and diarrhea (n = 1). Thus, three more patients were enrolled at 5 mg/m2, with no observed 
DLTs, establishing 5 mg/m2 as the recommended MTD. No additional grade 4 side effects were reported. 
Additional grade 3 effects at any time during trial therapy were ALT elevation (1 patient, 5mg/m2), reduced 
hemoglobin (2 patients at 3 mg/m2), infection (3 patients at 3 mg/m2), leucopenia (2 patients at 3mg/m2), 
mucositis (one patient), hypokalemia (1 patient at 3mg/m2, 1 patient at 5mg/m2), hyponatremia (1 patient at 
3mg/m2), anorexia (1 patient at 3 mg/m2), dizziness (1 patient at 3mg/m2), hyperglycemia (1 patient at 2.1 
mg/m2, 1 patient at 5 mg/m2),  nausea (1 patient at 3 mg/m2) and pain in the oral cavity (1 patient at 5 mg/m2). 
The adverse events seen were consistent with the known safety profile of everolimus in adults.38 
 
Everolimus pharmacokinetics in children were also found to be comparable to adults.  Everolimus was absorbed 
rapidly, with maximum concentrations achieved as early as 30 minutes after administration. The maximum 
everolimus whole-blood concentrations and AUC at each dose level were variable, but increased with dose.   
 
3.4.9 Phase I Study of Everolimus in Pediatric Patients with Refractory Solid Tumors 
 
Everolimus has been investigated in one phase I/II study (28 patients) in patients with TSC who have 
subependymal giant cell astrocytoma (SEGA) [CRAD001C2485]. Additionally, two phase III studies have been 
conducted in patients with TSC: study CRAD001M2301 in patients with TSC who have SEGA (117 patients) 
and study CRAD001M2302 in patients with renal angiomyolipoma (118 patients). CRAD001MIC02 is an 
ongoing, expanded access phase IIIb study for patients with TSC associated SEGA. Currently, there is a fourth 
phase III study CRAD001M2304 ongoing in which everolimus will be tested to evaluate whether its use as 
adjunctive treatment results in reducing seizure frequency in patients with TSC who have refractory seizures. 
 
Data on a small series of TSC patients who had systematic SEGAs which were treated with daily rapamycin 
therapy showed tumor size reduction by 46-63% within 2.5 to 5 months (Franz et al. 2006). All lesions exhibited 
regression and, in one case, necrosis. Interruption of therapy for one patient resulted in regrowth of the SEGA, 
but further regression of another 62-75% was seen upon the resumption of therapy. These results represented a 
new therapeutic paradigm for the treatment of SEGAs in the TSC patients with rapamycin and related agents.  
 
Therefore, in 2007, everolimus entered clinical development for TSC. Study C2485, conducted by Cincinnati 
Children’s Hospital Medical Center (CCHMC), is a prospective, non-randomized, open-label, investigator-
initiated, single-center 28-patient trial designed to evaluate the safety and efficacy of everolimus in patients ≥ 3 
years of age with SEGA associated with TSC. The primary efficacy endpoint was the change from baseline in 
volume of primary SEGA lesion at 6 months, as determined by central radiology review. At 6 months, 9 out of 28 
patients (32%, 95% CI: 16% to 52%) had a ≥ 50% reduction in the tumor volume of their largest SEGA lesion. 
The median duration of response for these 9 patients was 11.8 months (range 3.2 to 39.1 months). Response 
rate continues to improve as 58.3% of patients (14 of 23) who took everolimus for at least four years have 
experienced a reduction of ≥ 50% in the size of their largest SEGA relative to baseline. One patient has 
experienced a SEGA progression (defined as an increase of at least 25% to a value greater than baseline). The 
median time from first response to progression/censoring was 37 months (range: 6-63 months). No patient 
developed a new lesion and none required surgical resection or other therapy for SEGA. 
 
The safety and efficacy of everolimus in patients with TSC who have SEGA was studied further in 
[CRAD001M2301], a randomized (2:1), double-blind, placebo-controlled trial of everolimus conducted in 117 
pediatric and adult patients with SEGA and TSC. The main efficacy outcome measure was SEGA response rate 
based on independent central radiology review. After 6-months of study treatment, 35% of the patients treated 
with everolimus had at least a 50% reduction in SEGA volume compared to none in the placebo group (Franz et 
al 2013). Based on recent data, the response rate after at least 12-months of everolimus treatment has 
increased to 48%. SEGA progression was seen in nine of the all patients treated with everolimus (8.1%). In five 
patients, progression occurred after cessation of treatment or was associated with Cmin values which were 
markedly reduced or at zero. In one case of progression, the patient developed hydrocephalus that was 
successfully treated, in the absence of SEGA growth. In one case, progression reversed with further treatment. 
 
3.5 Study Rationale 
 

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Currently, no effective therapies for individuals with germline PTEN mutations exist to target neurocognitive and 
behavioral deficits, and effective interventions that target the core biologic alterations are crucial to optimize the 
short and long-term outcome of individuals affected with mutations in this gene.  
 
Like TSC, PTEN deficiency is an attractive target for treatment with mTOR inhibitors due the PTEN’s role 
through AKT to regulate specifically mTORC1, which is sensitive to rapamycin and analogs such as everolimus.   
 
Use of a neurocognitive composite score to determine efficacy has several advantages. Assuming that 
everolimus treatment does not impair some functions while improving others, a composite score is likely to be 
sensitive, highly reliable, can be computed in all patients even if 1 or 2 measurements are missing or could not 
be completed, and has good domain coverage to capture possible variability across patients in the specific 
neurocognitive domains showing improvement. An example of the latter advantage, is if some individuals show 
improvements in processing speed while others show improvements in working memory. The neurocognitive 
composite would identify improvements in both of these sets of patients. As such, the neurocognitive composite 
may be more sensitive to change than any individual measure. Furthermore, a composite primary efficacy 
outcome will not require additional multiple comparison correction. 
 
Exploratory measures were chosen based on their potential to serve as objective outcome measures in future 
trials. While none of these measures have specific evidence with PTEN mutation cases, they have some data 
regarding abnormalities within autism or developmental disability populations. In the cases of remote eye gaze 
tracking, resting EEG, and auditory evoked potentials, these measures are also reasonably inexpensive, can be 
acquired rapidly, and can be acquired in the majority of PTEN patients. 
 
4 Design and Methods 
 
4.1 Study Rationale 
 
This is a phase II, double-blind, randomized, parallel group, placebo-controlled, three center study evaluating 
treatment with everolimus versus placebo in 40 patients with PTEN gene mutations, ages 5-45 years (inclusive). 
It is expected that 60 patients will need to be screened to have 40 who will meet inclusion and exclusion criteria. 
 
There are four different phases in this study: pre-treatment (screening), a 6-month blinded treatment phase, a 6-
month open label phase for patients initially randomized to placebo, and a follow-up phase. Each of these 
phases is described in detail below. 
 
4.2 Pre-Treatment Phase (Screening) 
 
At screening, the investigator or his/her designee will assign a unique number to patients being considered for 
the study, as outlined in the 7904 Manual of Procedures. The patient/parent must provide a signed Informed 
Consent Form prior to any study screening evaluations being performed. Once the patient/parent provides a 
signed informed consent form and eligibility is confirmed (all inclusion/exclusion criteria have been 
verified), the investigator or his/her designee will register the patient for randomization.  
 
Screening evaluations will include demography, relevant medical history/current medical conditions, a physical 
examination (including a neurological examination), suicidal ideation/behavior assessment, vital signs, 
laboratory assessments, and other additional study entry evaluations. Primary endpoint neurocognitive data will 
be collected for screening purposes (SB-5, CPT-3, and Purdue Pegboard Test). Once the patient is confirmed 
as being eligible to be randomized, the baseline visit will be scheduled to occur within 6 weeks of the screening 
visit.  
 
Participants will be screened for signs and/or symptoms of cancer. If a patient presents at the screening visit 
with a mass that may be malignant or benign, the study physician will refer them to the appropriate health care 
providers. If the mass is benign, and the patient meets all other inclusion/exclusion criteria, the patient may be 
enrolled in the study. Additional information about cancer screening can be found in the Manual of Procedures. 
 
Participants will be screened for suicidal ideation and/or behaviors using the Columbia Suicide Severity Rating 
Scale (C-SSRS). Participants that are determined to have significant suicidal ideation and/or behaviors, as 

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defined in Section 4.6.5, will be ineligible to participate in the trial, and will be referred to the appropriate 
healthcare provider per the principal investigators discretion.  
 
A complete list of screening evaluations is provided in the schedule of evaluations. All of the above 
assessments/procedures must be conducted and evaluated prior to randomization. 
 
4.3 Blinded Treatment Phase 
 
All baseline procedures and evaluations should be completed within 6 weeks of the screening visit. If a patient’s 
safety lab results are out of range, and the investigator has reason to believe there were situational factors 
affecting the values, the screening labs may be repeated. The safety evaluations may be completed at a lab that 
is geographically close to the participant and either provide the results of that analysis directly to the study team, 
or a sign a release so the study team can obtain the results from the lab. If a patient’s baseline visit is more than 
6 weeks after the screening visit the participant must have the safety labs re-drawn and processed, have the 
physical and neurological exam re-done, and the study team will confirm that there are no changes in the 
medical history or changes to the patient’s current medications. If the screening safety evaluation and/or lab 
results are out-of-range, the participant will not be eligible to participate in the study. Patients who meet the 
study eligibility criteria following screening will be randomized to receive everolimus or placebo. The 
randomization ratio is 1:1, with one patient being randomly assigned to everolimus for every one patient 
randomly assigned to placebo. Treatment assignment via the RDCRN Members’ Website should occur prior to 
the Baseline visit to allow adequate time for pharmacy preparation. Patients must start treatment within 7 
days of the baseline visit, given that the participant still meets all inclusionary criteria and has not developed 
any exclusionary criteria. The medication should be taken at the same each day with a light, low-fat breakfast 
(i.e. 8:00 am with light breakfast).  
 
The duration of blinded phase this study is 6 months. Patients will have their first daily dose of everolimus or 
placebo within 7 days of Visit 2 (baseline) and will continue on treatment for 6 months unless an intolerable 
toxicity occurs, withdrawal of consent, or investigator decision to discontinue the patient from study treatment.   
 
Starting dose and adjustments are defined in Section. 6.2.  
 
Safety evaluations are routinely performed (visit 3/month 1, visit 5/month 3, and visit 8/month 6). Patients must 
be in a fasting state at the time of blood sampling for all laboratory evaluations including the lipid profile. 
Hematology and biochemistry assessments are required at the screening visit, the Month 1 visit, the Month 3 
visit, and the Month 6 visit. All blood samples obtained at each visit will be sent to LabConnect, LLC and 
associated testing facilities for analysis. If safety labs are out of range while the participant is randomized, a 
clinical assessment by the physician will take place that might include a repeat of blood work. Withdrawal from 
the study will be considered by the principal investigator if clinically indicated and after discussion with the study 
team. Complete details regarding all study required safety assessments are provided in Section 7.15. 
 
A PK sample will be collected at the Month 1 visit (one month ± 14 days from the baseline visit), when possible 
at steady-state, prior to dose administration during this visit, within 24 ± 4 hours after the last dose. Participants 
who receive a dose adjustment after this visit will work with study staff to get another PK sample taken at the 
study site or locally, by a designated and trained remote health care provider, 2 weeks (± 1 week) after the new 
dose has been taken. A PK sample may also be collected from a patient experiencing an adverse event or side 
effect. The decision to obtain this sample will be made by the site physician. 
 
Visits 4, 6, and 7 (months 2, 4, and 5) can be in-person, or by phone if that is preferred by the family. If 
performed by phone, a phone interview will be performed to collect safety and other data, review AEs, and 
assess changes in mood or behavior. All applicable CRFs will be completed. Modified visits 3, 5, 8 (months 1, 3, 
and 6) can also be conducted by phone/video conference if the family cannot make it to the study site within the 
designated window due to extenuating unforeseen circumstances.  
 
Tests that will be conducted during the blinded treatment phase include laboratory tests for safety, physical 
exams (including a neurological assessment), vital signs, and neuropsychological assessments. If unforeseen 
circumstances (i.e., unexpected personal reasons) prevent the patient from complying with the established visit 
schedule, the site can re-schedule the visit (within ± 14 days of the expected visit date). The reason(s) for any 
visit or treatment delays will be documented in the CRF for the appropriate visit. All on-site visits (screening, 

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baseline, month 1, month 3, and month 6) must be completed by the participant, and any missed visits would 
warrant termination from the study. A summary of the neuropsychological results may be made available at 
study completion.  
 
At the end of the double-blind phase (or early termination), the treatment code will be broken and the participant 
will be informed of their assigned treatment arm, using the process outlined in the 7904 Manual of Procedures. 
Participants who received the active compound will enter the follow-up phase of the study (section 4.5) and will 
be referred back to their treating physicians.  Individuals who were in the placebo group will be invited to enter 
Phase B, a 6-month open-label extension trial (section 4.4). The inclusion of the open-label phase may increase 
the acceptability of the project by families and increase their participation in the primary, controlled trial. All 
participants that received everolimus during the blinded treatment phase will enter the follow-up phase of the 
trial and will be referred back to their treating physicians.  A summary of patient’s participation in the study will 
be provided, if needed.  
 
 
4.4 Open Label Phase 
 
Individuals in the placebo group will be invited to enter the 6-month open-label extension trial that will follow the 
same schedule as the blinded treatment phase. The final visit, or the Month 6 visit, in the blinded treatment 
phase will serve as the baseline visit of the open label phase if the participant chooses to enter the open label 
phase. Safety labs obtained at the end of the blinded phase will be used as baseline safety labs for the open-
label phase. Similarly, the scores from the patient’s developmental assessments will be used as both the 6-
month blinded treatment phase data, and the baseline open-label phase data. Study staff should be prepared to 
dispense open label medication at this visit. If the patient is unable to enter the open-label phase at the final visit 
of the blinded treatment phase, the patient can come back within 2 weeks (+/- 2 weeks) of this visit date. 
Treatment, assessments, and outcomes of the open-label phase will also be identical to the double-blind phase. 
 
The next visit will be the Month 1 visit.  A PK sample, as well as specified safety labs, will be drawn on site and 
sent to LabConnect LLC. If the participant receives a dose adjustment at the time of this visit, study staff will 
work with the participant/family to get another sample taken at the study site or locally by a trained phlebotomist. 
The Month 3 visit and the Month 6 visit will include safety labs, developmental testing, optional EEG/AEP and 
eye tracking procedures, physical and neurological exams, vital signs, and other study questionnaires. A PK 
sample may also be collected from a patient experiencing an adverse event or side effect. The decision to 
obtain this sample will be made by the site physician. 
 
The Month 2, Month 4, and Month 5 visits can be in person or by-phone if that is preferred by the family. If 
performed by phone, a phone interview will be performed to collect safety and other data, review AEs, and 
assess changes in mood or behavior. All applicable CRFs will be completed.  
 
If unforeseen circumstances (i.e., unexpected personal reasons) prevent the patient from complying with the 
established visit schedule, the site can re-schedule the visit (within ± 14 days of the expected visit date). The 
reason(s) for any visit or treatment delays will be documented in the CRF for the appropriate visit. All on-site 
visits (month 1, month 3, and month 6) must be completed by the participant, and any missed visits would 
warrant termination from the study. Under extenuating unforeseen circumstances, visits 3, 5, 8 (months 1, 3, 
and 6) can be conducted by phone/video conference as a modified visit as to still capture obtainable data and 
maintain patient safety. A summary of the neuropsychological results may be made available at study 
completion. 
 
At the final visit of the open label phase, or the 6 month visit, participants will exit the trial. As done in the blinded 
treatment phase, they will receive a letter   
 
4.5 Follow-Up Phase 
 
All patients will have a follow-up phone call scheduled 28 days (± 14 days) after the last dose of the study 
treatment to follow for AEs and SAEs that may have occurred after discontinuation from the study treatment.  
See section 6.3.15 for additional details. 
 
4.6 Patient Selection and Inclusion/ Exclusion Criteria 

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Patients must have screening evaluations performed within the 6 weeks of the baseline visit and must still meet 
all inclusion and exclusion criteria at the time of the baseline visit. Results of all screening evaluations, which 
ensure that all inclusion and exclusion criteria have been satisfied, must be reviewed by the site’s Principal 
Investigator or his/her designee prior to enrollment of that patient. In addition, the patient must be thoroughly 
informed about all aspects of the study, including the study visit schedule and required evaluations and all 
regulatory requirements for informed consent. The written informed consent must be obtained from the patient 
prior to any screening activities. The following criteria apply to all patients enrolled onto the study, unless 
otherwise specified. 
 
4.6.1 Double-Blind Inclusion Criteria 
 
1. Male and female outpatients between 5 and 45 years of age (inclusive); 
2. Pathogenic PTEN mutation confirmed by clinical genetic testing; 
3.  Participant must be able to complete one of the following three standardized assessments: Conners’ 
Continuous Performance Tasks (CPT-3), Stanford Binet (SB-5; working memory), or the Purdue Pegboard 
Test;  
4. Performance below the age-adjusted population mean on at least one of the above standardized 
measures: attention (CPT-3, mean reaction time), working memory (SB5, working memory subscale), or 
fine motor skills (Purdue Pegboard Test; either dominant, non-dominant, or both hands); 
5. Adequate bone marrow function as shown by:  
a. Platelets ≥ 80,000/mm3 
b. Absolute neutrophil count ≥ 1,000/mm3  
c. Hemoglobin ≥ 9 g/dL; 
6. Adequate liver function as shown by: 
a. Total serum bilirubin <1.5 x ULN  
b. AST and ALT levels < 2.5 x ULN 
c. INR ≤2; 
7. Adequate renal function: serum creatinine < 1.5 x ULN; 
8. Signed informed consent obtained prior to any screening procedures; 
9. Individuals on psychotropic and anti-epileptic medications should maintain a stable dose for at least 2 
months prior to the screening visit; 
10. Negative serum pregnancy test for females at screening and no plans to become pregnant or conceive a 
child while participating in the study. The effects of mTOR inhibitors on the developing fetus at the doses 
used in this study are unknown. For this reason, women of child-bearing potential and men must agree to 
use adequate contraception prior to study entry and for the duration of the study. Estrogen-containing oral 
contraceptives are not recommended in women enrolled in this study. Abstinence or two effective non-
estrogen or barrier methods of contraception (such as condom + spermicidal foam) must be used;  
11. Medically stable with no active medical problem such as unstable seizure or cardiovascular disease as 
evidenced by a history; 
12. No anticipated changes in the frequency and intensity of existing interventions such as behavioral and 
developmental treatments, in home services, and speech therapy;  
13. No planned changes in school placement;  
14. For individuals under 18 or who are otherwise incapable, there must be an available caregiver who can 
reliably bring subject to clinic visits and provide trustworthy data; 
15. Able to communication fluently in English. 
4.6.2 Double-Blind Exclusion Criteria 
 
1. Patients currently receiving anticancer therapies or who have received anticancer therapies within 4 weeks 
of the start of Everolimus (including chemotherapy, radiation therapy, antibody based therapy, etc.); 
2. Known intolerance or hypersensitivity to Everolimus or other rapamycin analogs (e.g. sirolimus, 
temsirolimus); 
3. Known impairment of gastrointestinal (GI) function or GI disease that may significantly alter the absorption 
of oral Everolimus; 

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4. Uncontrolled diabetes mellitus as defined by HbA1c > 8% despite adequate therapy. Patients with a known 
history of impaired fasting glucose or diabetes mellitus (DM) may be included, however blood glucose and 
antidiabetic treatment must be monitored closely throughout the trial and adjusted as necessary; 
5. Patient with uncontrolled hyperlipidemia: fasting serum cholesterol > 300 mg/dL OR > 7.75 mmol/L AND 
fasting triglycerides > 2.5 x ULN.  
6. Patients who have any severe and/or uncontrolled medical or psychiatric conditions such as: 
a. Unstable angina pectoris, symptomatic congestive heart failure, myocardial infarction ≤6 months 
prior to start of Everolimus, serious uncontrolled cardiac arrhythmia, or any other clinically 
significant cardiac disease 
b. Symptomatic congestive heart failure of New York heart Association Class III or IV 
c. Active (acute or chronic) or uncontrolled severe infection, liver disease such as cirrhosis, 
decompensated liver disease, and active and chronic hepatitis (i.e. quantifiable HBV-DNA and/or 
positive HbsAg, quantifiable HCV-RNA), 
d. Known severely impaired lung function (spirometry and DLCO 50% or less of normal and O2 
saturation 88% or less at rest on room air)  
e. Active, bleeding diathesis; 
f. 
unstable angina pectoris, symptomatic congestive heart failure, myocardial infarction ≤6 months 
prior to start of Everolimus, serious uncontrolled cardiac arrhythmia, or any other clinically 
significant cardiac disease 
g. Symptomatic congestive heart failure of New York Heart Association Class III or Iactive (acute or 
chronic) or uncontrolled severe infection, liver disease such as cirrhosis, decompensated liver 
disease, and active and chronic hepatitis (i.e. quantifiable HBV-DNA and/or positive HbsAg, 
quantifiable HCV-RNA), 
h. known severely impaired lung function (spirometry and DLCO 50% or less of normal and O2 
saturation 88% or less at rest on room air), 
i. 
severe psychiatric disorders, such as bipolar disorders or schizophrenia, 
j. 
active substance-abuse, 
k. active, bleeding diathesis; 
7. Chronic treatment with corticosteroids or other immunosuppressive agents. Topical or inhaled 
corticosteroids are allowed; 
8. Known history of or seropositivity for Hepatitis B, Hepatitis C, or HIV 
9. Patients who have received live attenuated vaccines within 1 week of start of Everolimus and during the 
study. Patient should also avoid close contact with others who have received live attenuated vaccines. 
Examples of live attenuated vaccines include intranasal influenza, measles, mumps, rubella, oral polio, 
BCG, yellow fever, varicella and TY21a typhoid vaccines; 
10. Patients who have a history of another primary malignancy, with the exceptions of:  
a. non-melanoma skin cancer 
b. carcinoma in situ of the cervix, uteri, or breast from which the patient has been disease free for ≥3 
years; 
11. Planned changes to concomitant medications;   
12. Prior or concomitant therapy with known or possible anti-mTOR activity, including rapamycin (sirolimus);  
13. Concomitant therapy with strong inhibitor (e.g., cyclosporine and ketoconazole) or inducer of CYP3A; 
14.  Active infection at time of enrollment;  
15. Patients with a history of non-compliance to medical regimens or who are considered potentially unreliable 
or will not be able to complete the entire study; 
16. Patients who are currently part of or have participated in any clinical investigation with an investigational 
drug within 1 month prior to dosing; 
17. Pregnant or nursing (lactating) women; 
18. Women of child-bearing potential (WOCBP), defined as all women physiologically capable of becoming 
pregnant, must use highly effective methods of contraception during the study and 8 weeks after. Highly 
effective contraception methods include:  
a. A combination of any two of the following: 
i. 
Use of oral, injected or implanted hormonal non-estrogen containing methods of 
contraception or; 
ii. 
Placement of an intrauterine device (IUD) or intrauterine system (IUS); 
iii. 
Barrier methods of contraception: condom or occlusive cap (diaphragm or cervical/vault caps) 
with spermicidal foam/gel/film/cream/ vaginal suppository; 
b. Total abstinence or; 

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c. Male/female sterilization. 
Women are considered post-menopausal and not of child-bearing potential if they have had 12 
months or natural (spontaneous) amenorrhea with an appropriate clinical profile (e.g. age 
appropriate, history of vasomotor symptoms) or have had surgical bilateral oophorectomy (with 
or without hysterectomy) or tubal ligation at least six weeks prior to randomization. In the case 
of oophorectomy alone, only when the reproductive status of the woman has been confirmed by 
follow up hormone level assessment is she considered not of child-bearing potential. 
19. Male patients whose sexual partner(s) are WOCBP who are not willing to use adequate contraception, 
during the study and for 8 weeks after the end of treatment 
20. Major surgery, radiation therapy or stereotactic radio-surgery within previous 4 weeks at time of screening 
21. Neurosurgery within prior 6 months at time of screening. 
 
4.6.3 Open-Label Phase Inclusion Criteria 
 
1. Patients who completed Double-Blind phase of the study and were assigned to the placebo treatment 
arm; 
2. Verbal consent (and assent, as appropriate) obtained prior to any open-label phase study procedures. 
 
4.6.4 Screening for Hepatitis B, Hepatitis C, and HIV 
 
Study teams will review a list of risk factors for Hepatitis B, Hepatitis C, and HIV with participants/families. If the 
participant meets any of these risk factors, a serology blood sample will be drawn at the screening visit. If the 
participant is positive for any of the viruses, they will be ineligible to participate in the study. 
 
Patients that meet any of the risk factors below will have a blood sample drawn for a serology panel that will test 
for Hepatitis B (HBV-DNA, HBsAg, HBs Ab, and HBc Ab), Hepatitis C (quantitative RNA-PCR and HCV Ab), and 
HIV (HIV I/II).  
 
Currently live or have lived in Asia, Africa, Central and South America, Eastern Europe, Spain, Portugal, 
and Greece                                      
 
Had an operation in a developing country at any time 
 
Received a blood transfusion prior to 1990 
 
Are current or past IV drug user 
 
Have body piercing or tattoos 
 
Have a biological mother diagnosed with HIV 
 
Have a biological mother diagnosed with hepatitis B or hepatitis C 
 
Have a history of hepatitis B infection (e.g., dark urine, jaundice, right upper quadrant pain) or hepatitis 
C 
 
Have had household contact with hepatitis B or hepatitis C infected patient(s) 
 
Have current or prior high-risk sexual activity 
 
Have current or prior dialysis 
 
Hepatitis B, Hepatitis C, and HIV screening will be done at the discretion of the principal investigator at each 
site, using these factors. If a patient tests positive for HIV, the Principal Investigator at each enrolling site is 
responsible for reporting the result to the appropriate public health agency based on IRB and state regulations. 
The Principal Investigator will inform the patient or parent of the positive result and reporting plan. Patients that 
screen positive for HIV will be provided counseling. 
 
4.6.5 Columbia Suicide Severity Rating Scale (C-SSRS) 
 
In light of the recent evidence linking psychotropic medications to suicidal behaviors, it is important to closely 
monitor the mental well-being of patients taking medications that target the central nervous system61. Therefore, 
assessment of suicidal ideation/behaviors will be completed during the screening phase and will be monitored 
during the trial. The C-SSRS will be administered to assess the presence of suicidality throughout the entirety of 
the trial. 
 
The Columbia-Suicide Severity Rating Scale (C-SSRS) is an interview that prospectively assesses suicidal 
ideation and behavior using a semi-structured interview to probe patient responses. The C-SSRS is intended to 

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be used by individuals who have received training in its administration. Ultimately, the determination of the 
presence of suicidal ideation and/or behavior depends on the judgment of the individual administering the scale.  
 
A trained investigator will administer the C-SSRS. Patients 13 years of age and older will complete the scale for 
themselves. Parents/caregivers will complete the interview for patients that are under the age of 13, or for 
patients unable to understand and complete the assessment due to cognitive impairment. Additionally, the study 
physician will proactively assess patients for suicidality and mood disturbances through discussions with the 
patient and/or parent. Any positive responses will be further investigated, and appropriate medical treatment will 
be initiated. 
 
The C-SSRS will be completed at screening, Month 1, Month 3 and Month 6 of the blinded phase, and Month1, 
Month 3 and Month 6 of the open label phase. For the screening visit, the C-SSRS will be evaluate the last 12 
months. For Month 1, Month 3, and Month 6 of the double-blind and open-label phases, the C-SSRS will assess 
any changes from the previous visit. If, at any assessment, the Suicidal Ideation (questions #1-5) score is a 4 or 
above, or the patient (or parents) answers “yes” to any Suicidal Behavior item (actual attempt, interrupted 
attempt, aborted attempt, preparatory acts or behavior, or suicidal behavior), the patient must be referred to a 
health care professional for further assessment and/or treatment. If the participant meets any of these criteria at 
the screening visit, they will be excluded from the study. Once the participant has started study medication, the 
decision about whether the participant should be withdrawn from study treatment will be determined by the site 
PI, in collaboration with one of the site’s mental health professionals and Dr. Hardan. To note, any study 
investigator or research staff can refer a participant to the designated site mental health professional for any 
concerns about suicidality, even if the patient does not meet the criteria detailed above.  
 
The patient should not be allowed to leave the study center until the results of the C-SSRS are reviewed and the 
patient is not considered to be at risk. If there is doubt about whether a patient is at risk, the Investigator should 
obtain appropriate psychiatric consultation prior to releasing the patient. 
 
In the event of a suicide attempt or successful suicide, the investigator will complete a serious adverse events 
form.   
 
 
4.7 Early Termination 
 
If the participant, legal guardian, or the study physician make the decision to have the participant exit the study 
early, a follow-up visit must be scheduled 28 days (+/- 14 days) after termination. At this visit, the study 
physician should review the patient’s medical history, concomitant medications, and the DOTES. If the patient 
exited the trial due to an adverse event, this event should be reviewed and if it is still present the study team 
must follow-up with the patient/legal guardian for 56 days or until the event has subsided. At the completion of 
this follow-up visit, the participant may be unblinded and the results may be disclosed to the participant. 
 
 
5. Recruitment Methods 
 
Recruitment will leverage the observational study, The Natural History of Individuals with Autism and Germline 
Heterozygous PTEN mutations, and will occur at 3 sites: Cleveland Clinic, Stanford University, and Boston 
Children’s Hospital, with Cleveland Clinic expected to recruit at least 50% of the sample based on extensive 
experience with PTEN patients at this site. Over the last 5 years, we have recruited and comprehensively 
evaluated 35+ patients with PTEN ASD only from referrals to Cleveland Clinic and word of mouth. We anticipate 
that the majority of these individuals would participate in the proposed study.  
 
We will supplement our current recruitment efforts with several complementary strategies. First, Dr. Charis Eng 
(Cleveland Clinic) has developed a registry of > 400 individuals with PTEN mutations that includes > 50 PTEN 
cases with ASD or evidence of developmental delay. To date, this registry has not been tapped, but it will be 
used to recruit individuals for the proposed study. Dr. Eng’s lab also regularly screens individuals at risk of 
PTEN mutations using an online risk calculator and extensive referral network – growing her registry. Second, 
the two additional sites, Stanford University and Boston Children’s Hospital, also have experience with PTEN 
ASD patients and have an identified population that is interested in participating in this trial. Both of these sites 
will also leverage their relevant clinics to advertise and recruit for this study. Third, we have obtained support 

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from a community for caregivers of PTEN ASD patients (www.ptenlife.com) and the largest online group 
devoted to supporting families and patients affected with and by PHTS (www.ptenworld.com). Both groups have 
agreed to assist in recruitment. Finally, we will employ multiple advertising and recruiting tools both locally and 
nationally to raise awareness of the study.  
 
Potential subjects will be recruited through the same methods as listed above at the other participating sites. 
Recruitment will begin as soon as IRB approval is obtained. 
 
The study coordinator, principal investigator, co-investigators and sub-investigators at each site will be 
responsible for recruitment efforts.  An introductory letter to families with known diagnosis of PTEN and who 
otherwise meet eligibility criteria with an opt-out postcard as well as advertisements on advocacy group 
websites will be utilized for recruitment at each site. We will also utilize the Rare Diseases Clinical Research 
Network Developmental Synaptopathies Consortium (RDCRN DSC) contact registry, developed as a part of the 
natural history study, to inform individuals interested in research opportunities about the trial. This registry 
already has more than 80 distinct patients entered. 
 
5.1 Retention Strategies 
 
To retain participants throughout the 6-month trial, sites will use procedures that have proven successful in 
previous studies, such as phone calls to debrief after the most recent visit and phone calls or emails to prepare 
for the upcoming visit. Individual sites will seek IRB approval for each of the methods that they use prior to 
distribution. Whenever possible, study staff at each site should also try to schedule study visits on a day that is 
convenient for the family. Additionally, caregivers, or adult participants, will receive feedback on their child’s, or 
their, neurocognitive profile and changes over time at the end of the trial. Since there is a 50/50 chance of 
participants receiving everolimus or placebo, it is important for study staff to make sure the family is aware of the 
open-label phase of the study. Families should also be reminded that all travel expenses, throughout the entirety 
of the trial, will be covered. Finally, study staff should always express gratitude and thanks to both the patient 
and his/her family for participation in the study. 
 
6. Description of Study Treatments or Exposures/Predictors 
 
6.1 Everolimus Administration 
 
Patients will be randomized to receive either everolimus or placebo. Patients will be treated with blinded study 
treatment for 6 months, or until unacceptable toxicity or discontinuation for any other reason.  
 
For the duration of the trial, everolimus and placebo will be supplied to the research pharmacies at each site 
directly from Novartis. Everolimus and placebo will be formulated as identical tablets of 2.5mg or 5mg strength, 
blister-packed under aluminum foil in units of 10 tablets. Medication labels will comply with US legal 
requirements for investigational drug products and will be printed in English. The storage conditions for study 
drug will be described on the medication label. 
 
The study drug everolimus and/or placebo will be self-administered (by the patient or patient’s parent/guardian). 
The investigator should promote compliance by instructing the patient to take the study drug exactly as 
prescribed and by stating that compliance is necessary for the patient’s safety and the validity of the study. The 
patient should be instructed to contact the investigator if he/she is unable, for any reason, to take the study drug 
as prescribed. Everolimus should be administered orally once daily at the same time every day, consistently 
with a light, low-fat meal. Everolimus or placebo tablets should be opened only at the time of administration as 
drug is both hygroscopic and light-sensitive. The extent of absorption of everolimus through topical exposure is 
not known. Therefore, patients/caregivers are advised to avoid contact with the everolimus or placebo tablets 
and should wash their hands thoroughly before and after administration. 
 
For the Open-label phase: Everolimus is formulated as tablets for oral administration of 2.5mg, 5mg, and 10mg 
strength. Tablets are blister-packed under aluminum foil, which should be opened only at the time of 
administration as drug is both hygroscopic and light-sensitive. Refer to label for expiration date and storage 
conditions. 
 
6.2 Treatment Course Information 

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The average starting dose will be 4.5 mg/m2/day of trial therapy (either everolimus or placebo), rounded to the 
nearest 2.5mg dose. The BSA should be calculated (section 7.15) based on an accurate height and weight 
measurement performed according to institutional guidelines.  Leftover study medication, as well as all used 
blister packs, should be collected at each study visit, and drug should be accounted for at this time. If 2.5mg 
tablets become unavailable during the trial, patients prescribed a dose including 2.5mg tablets will be instructed 
to alternate between a higher and lower dose. The site physician will instruct the patient to take the higher dose 
on Monday, Wednesday, and Friday. The patient will take the lower dose on Tuesday, Thursday, Saturday, and 
Sunday. For example, a patient prescribed a dose of 7.5mg will take 10mg on Monday, Wednesday, and Friday, 
and 5mg on Tuesday, Thursday, Saturday, and Sunday. 
 
A maximum volume of 3 ml of blood will be drawn for trough everolimus PK levels when necessary. The blood 
draws will be timed to occur at 24 ± 4 hours after ingestion of the drug. Blood collection for PK samples may be 
conducted during a scheduled visit on-site or via a designated and trained remote healthcare service. 
Everolimus levels will be measured at ARUP laboratories in Salt Lake City, UT, in conjunction with LabConnect, 
LLC. When necessary, a kit for remote collection of blood samples can be sent to the participant and returned 
per the shipping instruction by express mail.  
 
Dose adjustments in the blinded phase of the trial can occur in both placebo (randomized modification) and 
active (based on PK findings) arms and will be at the discretion of a central unblinded investigator, Sarah 
Spence, M.D. or back-up unblinded investigator, Kiran Maski, M.D. In the event both Drs. Spence and Maski are 
unavailable, Dr. David Franz, Medical Review Officer, will serve as the unblinded investigator for the purposes 
of PK-driven dose adjustments. An unblinded research nurse will assist the unblinded physicians and the 
Medical Review Officer in their roles and will make sure that communications between them and the different 
sites is occurring in a timely manner. 
 
Dose adjustments will be permitted, based on safety findings and PK levels. The site PI will initiate any safety 
level related dose adjustments according to the protocol, in collaboration with the Medical Review Officer as 
appropriate. PK based dose adjustments will be initiated by the unblinded physician. All dose adjustments will 
be made using the 2.5mg tablets (i.e. increase by 2.5mg, decrease by 2.5mg, or maintain dose). If a dose 
adjustment is made for safety purposes, the unblinded physicians and Medical Review Officer should be made 
aware of any dose modifications as soon as possible.  
 
Sites may substitute 5mg tablets (or 10mg tablets in open label) for 2.5mg tablets where necessary, as long as 
the single dose level increase or decrease is maintained. For example, an adjustment from 7.5mg to 10mg can 
be made by prescribing two 5mg tablets (or one 10mg tablet in open label) rather than one 5mg and two 2.5mg 
tablets. Following a dose adjustment, sites can mail the extra 5mg tablets to the participant in between visits to 
make up for the remaining days until the next site visit. One level dose increase would be increasing the dose by 
2.5mg and a one level dose decrease would be decreasing the dose by 2.5mg as stated above. 
 
Placebo Arm: Placebo dose adjustment procedures are defined in the unblinded dose adjustment protocol – 
accessible only to the unblinded physician(s) and the DMCC. The site team will be blinded with respect to 
treatment assignment at all times. Dr. Maski, Dr. Spence, and Dr. Franz will receive instructions regarding dose 
modifications for participants on placebo from the DMCC. When PK values become available for individuals in 
placebo arm, Dr. Maski or Dr. Spence will refer to the provided instructions and notify site PI/coordinator of dose 
status (increase, decrease, or maintain) for individuals assigned to a dose adjustment.  
 
Active Treatment Arm: Based on the everolimus PK level, Dr. Maski, Dr. Spence, or Dr. Franz will determine 
whether a dose adjustment is warranted, i.e. the value falls outside the range of 5-15 ng/ml. The unblinded 
physician(s) will notify the local research team and PI how to proceed with next dose (increase by 2.5mg, 
decrease by 2.5mg, or maintain dose). Following any dose adjustments, a blood sample will be redrawn two 
weeks (+/- one week) after the modification. The unblinded physician will assess the everolimus PK level and 
notify the local study team to maintain or adjust the dose accordingly to achieve the target range of 5-15ng/ml. 
This process will be repeated until the everolimus level falls within the target range.  
 
Dose adjustments in the open label phase of the study will be made by the site principal investigator. The PK 
value taken at the Month 1 visit will be released to the site physician by the unblinded coordinator and dose 
adjustments will be made based on the value.  

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A detailed explanation of other permitted dose adjustments can be found in Section 7.7.3.   
 
6.3 Known Undesirable Effects of Study Drug/Treatment  
 
Overall, safety data available from completed, controlled and uncontrolled studies indicate that everolimus is 
generally well tolerated at weekly or daily doses. The safety profile is characterized by manageable adverse 
events (AEs). These AEs are generally reversible and non-cumulative.  
 
Adverse events most frequently observed with everolimus are stomatitis, mouth ulcers, acne, infection, and 
menstrual irregularities. Adverse events that are also common include: rash, diarrhea, fatigue, asthenia, nausea, 
peripheral edema, decreased appetite, headache, dysgeusia, epistaxis, mucosal inflammation, pneumonitis, 
weight decreased, vomiting, pruritus, cough, dyspnea, dry skin, nail disorder, aggression, and pyrexia.  
 
Overall, the most frequently observed laboratory abnormalities include: decreased hematology parameters 
including hemoglobin, lymphocytes, platelets, and neutrophils; increased clinical chemistry parameters including 
cholesterol, triglycerides, glucose, aspartate transaminases, creatinine, alanine transaminases, and bilirubin; 
and decreased clinical chemistry parameters including phosphate and potassium.  
 
The majority of these AEs have been of mild to moderate severity (NCI CTC grade 1-2). Recommendations for 
dose adjustments, should any of these treatment related adverse events occur, are given in Table 3.  
 
6.3.1 Adverse Drug Reactions (ADRs) Reported for Previous Tuberous Sclerosis Complex Studies  
 
The most frequent ADRs (incidence ≥ 1/10 and suspected to be related to treatment by the investigator) from 
the pooled safety database are (in decreasing order): stomatitis, amenorrhea, upper respiratory tract infections, 
hypercholesterolemia, nasopharyngitis, acne, menstruation irregular, sinusitis, and pneumonia 
The most frequent grade 3/4 adverse reactions (incidence ≥ 1/100 to < 1/10 and suspected to be related to 
treatment by the investigator) were stomatitis, amenorrhea, pneumonia, neutropenia, pyrexia, and 
gastroenteritis viral. 
 
The below table shows the incidence of ADRs based on pooled data in patients receiving everolimus in the TSC 
studies (including both the double-blind and open-label study and extension periods). ADRs are listed according 
to MedDRA system organ class. Frequency categories are defined using the following convention: very common 
(≥ 1/10); common (≥ 1/100 to < 1/10); uncommon (≥ 1/1,000 to < 1/100); rare (≥ 1/10,000 to < 1/1,000); very 
rare (< 1/10,000); not known (cannot be estimated from the available data). Within each frequency grouping, 
ADRs are presented in order of decreasing frequency. 
 
Infections and infestations 
Very common  
- Upper respiratory tract infection, nasopharngitis, sinusitis, pneumonia 
Common        
- Otitis media, urinary tract infection, pharyngitis, cellulitis, 
                          
   pharyngitis streptococcal, gastroenteritis viral, gingivitis 
Uncommon     
- Herpes zoster, bronchitis viral 
 
Blood and lymphatic system disorders 
Common      
- Neutropenia, anemia, leukopenia, lymphopenia, thrombocytopenia 
 
Immune system disorders 
Uncommon     
- Hypersensitivity 
 
Metabolism and nutrition disorders 
Very common   
- Hypercholesterolemia 
Common         
- Hyperlipidemia, decreased appetite, hypophosphatemia, hypertriglyceridemia 
 
Psychiatric disorders 
Common          
- Insomnia 
Uncommon      
- Aggression 
 

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Nervous system disorders 
Common          
- Headache, dysgeusia 
 
Vascular disorders 
Common        
- Hypertension, lymphedema 
 
Respiratory, thoracic and mediastinal disorders 
Common         
- Cough, epistaxis 
Uncommon      
- Pneumonitis 
 
Gastrointestinal disorders 
Very common 
- Stomatitis 
Common        
- Diarrhea, nausea, vomiting, abdominal pain, oral pain, flatulence, constipation, 
gastritis 
 
Skin and subcutaneous tissue disorders 
Very Common 
- Acne 
Common         
- Rash, dermatitis acneiform, dry skin 
Uncommon     
- Angioedema 
 
Renal and urinary disorders 
Common           
- Proteinuria 
 
Reproductive system and breast disorders 
Very Common   
- Amenorrhea C, menstruation irregular C, 
Common           
- Menorrhagia, vaginal hemorrhage, ovarian cyst, menstruation delayed C                                   
 
General disorders and administration site conditions 
Common           
- Fatigue, pyrexia, irritability 
 
Investigations 
Common           
- Blood lactate dehydrogenase increased, blood luteinizing hormone increased 
Uncommon     
- Blood follicle stimulating hormone increased 
 
Includes very common: stomatitis, mouth ulcerationaphthous stomatitis; uncommon: gingival pain, glossitis, lip 
ulceration.  
 
Includes common: rash, rash erythematous. 
 
Includes uncommon: erythema, rash macular, rash maculo-papular, rash generalized. 
 
Frequency is based upon number of women age 10 to 55 years of age in the safety pool. 
 
6.3.2 Management of Infections  
 
Everolimus has immunosuppressant properties and may predispose patients to bacterial, fungal, viral or 
protozoal infections, including infections with opportunistic pathogens. Patients taking everolimus are therefore 
at an increased risk of infection. In oncology patients, some infections have been severe, and rarely have had a 
fatal outcome. Physicians should be aware of the increased risk of infection, and should warn patients and their 
caregivers to be vigilant for signs and symptoms of infection, and to seek medical attention immediately should 
such signs or symptoms occur. Treat pre-existing infections prior to starting treatment with everolimus. Should 
an infection occur, anti-infective should be prescribed as clinically appropriate, and in the case of clinically 
significant infection, consideration should be given to withholding study medication until resolution of the 
infection. 
 
As everolimus is an immunosuppressant, live vaccinations should be avoided (as recommended by the Center 
for Disease Control) (FluMist®, MMR, Varicella, human papillomavirus or HPV, rotavirus or rotarix vaccine, 
smallpox or vaccinia, rabies vaccine, yellow fever, typhoid Ty21a and BCG).  It is okay and recommended for 

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participants to receive a flu shot every year (unless contraindicated) as that is an inactivated vaccine. This 
precaution pertains to the risk for inadequate antibody response more than risk for disease from the 
immunization. 
 
6.3.3 Management of Skin Toxicity  
 
For patients with grade 1 toxicity, no specific supportive care is usually needed or indicated. Rash must be 
reported as an AE. Patients with grade 2 or higher toxicity may be treated with the following suggested 
supportive measures at the discretion of the investigator: oral minocycline, topical tetracycline, topical 
clindamycin, topical silver sulfadiazine, diphenhydramine, oral prednisolone (short course), topical 
corticosteroids, or pimecrolimus.  
 
6.3.4 Management of Hypersensitivity Reactions  
 
Hypersensitivity reactions manifested by symptoms including, but not limited to, anaphylaxis, dyspnea, flushing, 
chest pain or angioedema (e.g. swelling of the airways or tongue, with or without respiratory impairment) have 
been observed with everolimus.  
 
6.3.5 Angioedema with Concomitant Use of Angiotensin-Converting Enzyme (ACE) Inhibitors 
 
Patients taking concomitant ACE inhibitor therapy may be at increased risk for angioedema.  
 
6.3.6 Renal Failure Events 
 
Cases of renal failure (including acute renal failure), some with fatal outcome, occurred in patients treated with 
everolimus. Renal function of patients should be monitored particularly where patients have additional risk 
factors that may further impair renal function. Elevations of serum creatinine, usually mild, and proteinuria have 
been reported in patients taking everolimus. Monitoring of renal function, including measurement of blood urea 
nitrogen (BUN), urinary protein, or serum creatinine, is recommended prior to the start of everolimus therapy 
and periodically after.  
 
6.3.7 Management of Mouth Ulcers/Stomatitis/Oral Mucositis  
 
Stomatitis/oral mucositis/mouth ulcers due to everolimus should be treated using appropriate locally available 
supportive care. Please note that investigators in earlier trials have described the oral toxicities associated with 
everolimus as mouth ulcers, rather than mucositis or stomatitis. If your examination reveals mouth ulcers rather 
than a more general inflammation of the mouth, please classify the adverse event as such. Please follow the 
paradigm below for treatment of stomatitis/oral mucositis/mouth ulcers: 
 
1. For mild toxicity (grade 1), use conservative measures such as non-alcoholic mouth wash or salt water 
(0.9%) mouth wash several times a day until resolution. 
2. For more severe toxicity (grade 2 in which case patients have pain but are able to maintain adequate oral 
alimentation, or grade 3 in which case patients cannot maintain adequate oral alimentation), the suggested 
treatments are topical analgesic mouth treatments (i.e., local anesthetics such as benzocaine, butyl 
aminobenzoate, tetracaine hydrochloride, menthol, or phenol) with or without topical corticosteroids, 
such as triamcinolone oral paste 0.1% (Kenalog in Orabase®). 
3. Agents containing hydrogen peroxide, iodine, and thyme derivatives may tend to worsen mouth ulcers. It is 
preferable to avoid these agents. 
4. Antifungal agents must be avoided unless a fungal infection is diagnosed. In particular, systemic imidazole 
antifungal agents (ketoconazole, fluconazole, itraconazole, etc.) should be avoided in all patients due to 
their strong inhibition of everolimus metabolism, therefore leading to higher everolimus exposures. 
Therefore, topical antifungal agents are preferred if an infection is diagnosed. Similarly, antiviral agents such 
as acyclovir should be avoided unless a viral infection is diagnosed. 
 
For additional details about the treatment of stomatitis as an everolimus adverse drug reaction (ADR), see the 
7904 Study Manual of Procedures. 
 

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6.3.8 Management of Hyperlipidemia and Hyperglycemia  
 
Management of hyperlipidemia should take into account the pre-treatment status and dietary habits of the 
patient. Blood tests to monitor hyperlipidemia must be taken in the fasting state. Hyperlipidemia and 
hypertriglyceridemia should be treated according to local best clinical practice. Grade 3 hypercholesterolemia (> 
400 mg/dL or 10.34 mmol/L) or grade 3 hypertriglyceridemia (>5 × ULN) should be treated as clinically indicated 
with a 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitor (e.g. atorvastatin, pravastatin) or appropriate 
lipid-lowering medication, in addition to diet. Patients should be monitored clinically and through serum 
biochemistry for the development of rhabdomyolysis and other adverse events as required in the product 
label/data sheets for HMG-CoA reductase inhibitors. 
 
Note: Concomitant therapy with fibrates and an HMG-CoA reductase inhibitor is associated with an increased 
risk of a rare but serious skeletal muscle toxicity manifested by rhabdomyolysis, markedly elevated creatine 
phosphokinase (CPK) levels and myoglobinuria, acute renal failure and sometimes death. The risk versus 
benefit of using this therapy should be determined for individual patients based on their risk of cardiovascular 
complications of hyperlipidemia. 
 
Grade 3 hyperglycemia has been observed in patients receiving everolimus therapy. In almost all cases the 
affected patients had an abnormal fasting glucose at baseline. Based on this finding, it is suggested that optimal 
glucose control should be achieved before starting a patient on study drug, and that glucose control should be 
monitored during the trial. More frequent monitoring is recommended when everolimus is co-administered with 
other drugs that may induce hyperglycemia. 
 
6.3.9 Management of Diarrhea  
 
Diarrhea attributed to everolimus toxicity may be treated with supportive care, such as loperamide, initiated at 
earliest onset (for example, 4 mg orally followed by 2 mg orally every 2 hours until resolution of diarrhea). Other 
medications for diarrhea may be used as needed. 
 
6.3.10 Dosing Modifications In Case of Treatment Related Toxicities 
 
Table 3 Criteria for dose modification/re-initiation in case of toxicity suspected to be related to study treatment 
Toxicity 
Actions 
Pneumonitis 
See Table  5 
Hyperlipidemia and/or 
hypertriglyceridemia 
Any grade: 
Treat according to best clinical practice. No specific dose reductions 
are needed. 
Hyperglycemia 
Any grade: 
Treat according to best clinical practice. No specific dose reductions 
are needed.  
Stomatitis 
Grade 2: 
Interrupt study drug until resolution to ≤ grade 1. Restart at same 
dose. 
 
Grade 3: 
Interrupt study drug until resolution to ≤ grade 1. Reintroduce study 
drug at the next lower dose level**. 
Discontinue study drug if stomatitis does not recover to ≤grade 1 
within 4 weeks. 
 
Grade 4: 
Discontinue study drug 
 

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Other toxicities 
Grade 2 and 3: 
Interrupt administration until resolution to ≤grade 1. Restart at same 
dose. 
 
Grade 4: 
Hold study drug until recovery to ≤ grade 1. Reintroduce study drug at 
the lower dose level**, if available. 
 
Toxicity requiring interruption for > 6 
weeks 
 
Permanently discontinue treatment. 
No specific dose adjustments are recommended for Grade 1 toxicity. However, physicians should always 
manage patients according to their medical judgment based on the particular clinical circumstances. 
**To determine the next lowest dose level, please refer to Table. 5. 
 
Table 4 Dose modification guidelines for hematologic toxicities 
Toxicity 
Actions 
Thrombocytopenia 
Platelet Count 
 
≥ 75000/mm3: No change 
 
50000/mm3 to 75000/mm3: Hold study drug until recovery to ≥ 
75000/mm3. Reintroduce study drug at the same dose level  
 
< 50000/ mm3: Hold study drug until recovery to ≥ 
75000/mm3. Reintroduce everolimus at the next lower dose 
level, if available 
 
Absolute Neutrophil Count (ANC) 
 
≥ 1000/ mm3: No change 
 
500/mm3 to 1000/ mm3: Hold study drug until recovery to ≥ 
1000/mm3. Reintroduce study drug at the same dose level  
 
< 500/ mm3: Hold until recovery to ≥ 1000/ mm3. Reintroduce 
everolimus at the next lower dose level, if available. 
 
Febrile Neutropenia 
Hold further dosing until ANC ≥ 1250/mm3 and no fever. 
Then resume dosing at the next lower dose level** if available. 
 
Toxicity requiring interruption for >6 
weeks 
 
Permanently discontinue treatment. 
Physicians should always manage patients according to their medical judgment based on the particular 
clinical circumstances. 
 
6.3.11 Management of Non-Infectious Pneumonitis 
 
Pneumonitis is a recognized adverse effect of rapamycins (sirolimus, temsirolimus, everolimus). Numerous case 
reports in the literature suggest that rapamycin-associated pneumonitis is relatively non-aggressive, limited in 
extent and reversible upon drug discontinuation. In the largest completed phase III trial using everolimus in 
oncology (in patients with metastatic renal cell carcinoma), the frequency of clinically apparent pneumonitis was 
14%. 4% of cases were CTC grade 3, and 0 were CTC grade 4. To date, three cases of pneumonitis have had a 
fatal outcome in more than 4,000 adult oncology patients treated with everolimus. All three cases were 
associated with overwhelming systemic infection or disease progression. 
 
Individuals participating in this trial will be questioned at each study visit as to the presence of new or changed 
pulmonary symptoms consistent with lung toxicity. If an investigator suspects a patient may be developing 
pneumonitis, the patient should be managed according to Table 5. Investigations such as pulmonary function 
tests, CT chest and referral to a pulmonologist should be considered. 
 
  Table 5 
Management of non-infectious pneumonitis  

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Worst Grade 
Pneumonitis 
Required Investigations 
Management 
of 
Pneumonitis 
Study Treatment Dose Adjustment 
Grade 1 
CT scans with lung windows 
and pulmonary function testing 
including: spirometry, DLCO, and 
room air O2 saturation at rest. 
Repeat CT scan at least every 
12 weeks until return to within 
normal limits. 
No 
specific 
therapy is required 
Administer 100% of study treatment 
dose. 
Grade 2 
CT scan with lung windows.  
Consider pulmonary function 
testing including: spirometry, 
DLCO, 
and 
room 
air 
O2 
saturation at rest. Repeat CT 
scan at least every 4-12 weeks 
until return to within normal 
limits. Consider bronchoscopy 
with biopsy and/or BAL 
Symptomatic only. 
Consider 
corticosteroids 
if 
symptoms 
are 
troublesome. 
Reduce study treatment dose by 1 
dose level until recovery to < Grade 
1. Study treatment may also be 
interrupted 
if 
symptoms 
are 
troublesome. 
Patients 
will 
discontinue study treatment if they 
fail to recover to < Grade 1 within 3 
weeks. 
Grade 3 
CT scan with lung windows and 
pulmonary 
function 
testing 
including spirometry, DLCO, and 
room air O2 saturation at rest. 
Repeat at least every 4-8 
weeks until return to within 
normal 
limits. 
Bronchoscopy 
with biopsy and/or BAL is 
recommended. 
Consider 
corticosteroids 
if 
infective origin is 
ruled out. Taper 
as 
medically 
indicated. 
Hold treatment until recovery to < 
Grade 1. May restart study treatment 
within 3 weeks at a reduced dose 
(by one level**) if evidence of clinical 
benefit. 
Grade 4 
CT scan with lung windows and 
required 
pulmonary 
function 
testing 
including 
spirometry, 
DLCO, 
and 
room 
air 
O2 
saturation at rest. Repeat at 
least every 4-8 weeks until 
return to within normal limits. 
Bronchoscopy 
with 
biopsy 
and/or BAL is recommended if 
possible.  
Consider 
corticosteroids 
if 
infective origin is 
ruled out. Taper 
as 
medically 
indicated. 
Discontinue treatment. 
 
 
 
6.3.12 Follow-Up for Toxicities 
 
Patients whose treatment is interrupted or permanently discontinued due to an adverse event or abnormal 
laboratory value suspected to be related to study treatment must be followed at least weekly until the adverse 
event or abnormal laboratory resolves or returns to grade 1. If a patient requires a dose delay of ≥ 6 weeks from 
the intended day of the next scheduled dose, then the patient must be discontinued from the study. 
 
All patients will be followed for adverse events and serious adverse events for 28 days following the last dose of 
study drug. Beyond these 28 days, any serious adverse events that are suspected to be related to the study 
drug that are ongoing at the follow-up call will continue to be monitored for an additional next 8 weeks (56 days) 
or until the event resolves. Any changes in medication/therapy given during the follow-up period of 28 days will 
be recorded on the CRF. 
 
7. Description of Study Procedures 
 
7.1 Written Informed Consent 
 

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Written informed consent will be obtained from each participant before any study-specific procedures or 
assessments are done and after the aims, methods, anticipated benefits, and potential hazards are explained.  
The participant’s willingness to participate in the study will be documented in writing in a consent form, which will 
be signed by the participant with the date of that signature indicated.  The investigator will keep the original 
consent forms and signed copies will be given to the participants.  It will also be explained to the participants 
that they are free to refuse entry into the study and free to withdraw from the study at any time without prejudice 
to future treatment.  
 
7.2 Process of Consent 
 
Informed consent is an ongoing process that includes the signing of an informed consent document and, when 
applicable, an assent form. Parents/guardians/subjects are required to sign an informed consent prior to being 
screened, and before undergoing any study procedures or assessments, in accordance with International 
Conference on Harmonization (ICH) E6; 4.8, “Informed Consent of Trial Subjects.” When substantial 
modifications are made to the informed consent, the IRB may require that all subjects currently enrolled in the 
study will be re-consented; ICH E6; 4.8 guidelines would still apply.  The informed consent process will be 
documented by the study site within the subjects’ research records.  
 
Parents/guardians/subjects will be provided with a copy of the informed consent, and assent form (if applicable), 
that explains the purpose of the study, the study procedures, and assessments.  Parents/guardians/subjects will 
also be provided with the contact information of the investigator and qualified personnel who can assist with 
their questions and concerns. 
 
7.3 Remote Consent Process 
 
Remote consenting procedures will be used for re-consenting participants that are no longer coming to the 
research site for study related visits. Consenting by remote process should not be used for initial consent, nor 
for participants actively attending study visits in-clinic. Study staff will use secure methods to communicate and 
transmit information, such as scanning, faxing, or mailing the documents, each participant or their 
parent(s)/guardian(s) will be provided with two blank copies of the informed consent/assent documents: one to 
keep and one to return.  Once the participant/parent/guardian has received the consent documents, an 
authorized research staff member will explain the study and review the consent document, highlighting the 
changes that have been made to this consent form version, with each participant by phone or videoconference.  
If assent is required, the authorized staff must have a discussion with both parent/legal guardian and child 
participant.  
 
Once all questions have been answered and the authorized staff member feels confident that each 
participant/parent/guardian understands the study and the changes, the participant (adult) and/or a 
parent/guardian (child) will sign and date the consent form. The signed consent form will be returned to the site 
via email, fax, or mail.  If there is a separate assent form, the participant will need to sign, date, and return the 
assent with the consent form.  When the signed consent form is returned to the study team, the staff member 
who provided the explanation of the study will sign the appropriate signature line, using the current date. Please 
note, the site signature date should not be back-dated to coincide with the participant’s signature. 
 
It is recommended that site staff document that consent was obtained remotely, via phone or videoconference, 
with the current date and date the form(s) were mailed/emailed/faxed back. This can be accomplished with a 
note under the PI signature line on the consent form. Example: “Discussed with [patient name and 
parent/guardian name] via telephone/videoconference on [insert date], and received sign consent form on [insert 
date].” 
 
Authorized staff should document the entire informed consent/assent process for each participant in a memo-to-
file. 
 
7.4 Patient Numbering 
 
Each patient in the study will be uniquely identified.  A Local ID number should be assigned as described in the 
7904 Manual of Procedures.  
 

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When the patient has signed the informed consent form, the investigator or his/her staff will log onto the 
electronic data capture system and provide the requested identifying information including the Local ID number 
for the patient. Participants will maintain the same Local ID for both the Double Blind and Open Label phases, 
as applicable. 
 
7.5 Randomization Procedure 
 
Subjects will be randomized 1:1 to everolimus or placebo after meeting all eligibility criteria. A designated 
member of the research team will communicate to the participant that randomization has occurred and sufficient 
study drug will be dispensed to cover the interval until the next study visit. This is a double-blind study. The 
study design allows patient unblinding during the double-blind phase only in very precise circumstances (section 
7.6.1). 
 
Randomization data are to be kept strictly confidential until the time of unblinding at discontinuation or at the 
time of final analysis. These data will not be accessible to anyone involved in the conduct of the study with the 
exception of the unblinded physicians, the Medical Monitor, the Data Safety and Monitoring Committee (DSMC), 
and the Data Management Coordinating Center (DMCC). The DSMC will review safety data as outlined in 
section 15. The identity of treatment arms will be concealed by the use of study drugs (everolimus and matching 
placebo) that are identical in packaging, labeling, schedule of administration and in appearance.   
 
Prior to Baseline (Visit 2), the investigator or his/her designee will log onto the data system to verify that the 
patient fulfills all eligibility criteria and randomize the patient to a treatment arm. The system will assign a 
randomization arm, as described in the 7904 Manual of Procedures. The pharmacy will be responsible for 
dispensing the active drug or placebo based on the procedures outlined in the 7904 Pharmacy Manual of 
Procedures. The randomization will be stratified by age. Age will be stratified by two levels. Level 1 will be 
participants aged 5-21 years old, and level 2 will be 22-45 year olds. Permuted block sizes of two and four will 
be used. At the conclusion of the 6-month double-blind phase, the blind will be broken immediately. Individuals 
on the active drug will exit the study, and those who were on placebo will be invited to join the open label phase.  
 
7.6 Double-Blind Treatment Assignment Unblinding 
 
Prior to the Month 6 visit or early termination, the site will obtain the unblinded treatment assignment via the 
procedures described in the 7904 Manual of Procedures. At the conclusion of the double-blind phase or early 
termination, the blind will be broken immediately. Individuals on the active drug will exit the study, and those 
who were on placebo will be invited to join the open label phase (section 4.4). 
 
7.6.1 Emergency Unblinding Treatment Assignment 
 
In general, circumstances that might lead to emergency unblinding are rare. Most often, study drug 
discontinuation and knowledge of the possible treatment assignments are sufficient to treat a study patient who 
presents with an emergency condition. One unusual circumstance in which unblinding might be necessary is 
when a patient requires emergency surgery and the anesthesiologist needs to know all medications that the 
patient has been exposed to in order to make proper decisions about treatment and support during the surgery.  
 
Emergency unblinding should only be done when necessary in order to treat the patient. Emergency code 
breaks are performed using the database, as described in the 7904 Manual of Procedures.  
 
It is the investigator’s responsibility to ensure that there is a procedure in place for emergency unblinding. The 
investigator will inform the patient how to contact his/her backup in cases of emergency when he/she is 
unavailable.  
 
Study drug must be discontinued after emergency unblinding. The investigator is not allowed to place 
emergency unblinded patients into open-label everolimus therapy.  
 
7.7 Treating the Patient  
 
7.7.1 Study Drug Administration 
 

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Everolimus or placebo will be dispensed by the study center personnel on an outpatient basis. Patients will be 
provided with study drug at Visit 2 (Baseline), and each subsequent on-site visit for self-administration at home.  
 
The first dose of everolimus or placebo must be taken within 7 days of Visit 2 (Baseline). Patients will be 
instructed to take everolimus or placebo by mouth regularly, with the dose being taken at the same time in the 
morning every day. Everolimus or placebo tablets should be swallowed whole with a glass of water. The tablets 
should not be chewed or crushed. Any dietary habits around the time of everolimus or matching placebo intake 
should be as consistent as possible throughout the study. The extent of absorption of everolimus through topical 
exposure is not known. Therefore, patients/caregivers are advised to avoid contact with the everolimus or 
placebo tablets and should wear gloves and should wash their hands thoroughly before and after administration.  
If vomiting occurs, no attempt should be made to replace the vomited dose. Patients should be instructed that if 
they miss a dose one day, they must not take any extra dose the next day.  
 
Patients should be requested to bring their unused study drug, including the empty blister packs, to the clinic at 
each visit.  
 
Patients will receive treatment with study drug for 6 months or until the occurrence of unacceptable toxicity or 
the investigator or patient decides that continuation is not in the best interest of the patient. Interruption for 
toxicity should follow the instructions in Table 3. 
 
7.7.2 Treatment Compliance 
 
Compliance should be verified by the investigator’s staff through counting the number of tablets consumed 
between visits. Compliance will be assessed by the investigator or his/her designee at each visit using pill 
counts and review of the drug diary. The site must maintain an overall drug accountability log for the study, as 
well as individual accountability records for each patient.  The dose, amount dispensed, amount taken, and 
amount remaining unused must be recorded in the source document. The patient will return all unused study 
drug at each dispensing visit, except Visit 2/Baseline, and at the End of Study visit.  
 
Patients will be requested to bring their unused medication including empty packaging to the clinic at each 
visit. 
 
All doses taken by the patient and all dose changes during the study must be recorded on the Dosage 
Administration Record CRF and the Dose Adjustment Log CRF, respectively.  
 
The investigator or his/her designee must keep documentation (overall drug accountability log for the study 
as well as individual study drug accountability records for each patient) of tablets administered, tablets used, 
dose changes, dates dispensed and intervals between visits.    
 
7.7.3 Other Permitted Study Drug Adjustments 
 
Standard dosing modifications will be made as described in Section 6.2.  
 
For patients who are unable to tolerate the protocol-specified dosing schedule, dose adjustments are permitted, 
as described in Tables 3, 4, and 5, in order to keep the patient on study drug. In addition, if any surgery is 
planned, trial therapy should be interrupted one week prior to surgery and should be re-started as soon as 
possible after wound healing. 
 
Dose modifications initiated by the site investigator (such as in response to a toxicity or safety lab level) will not 
require an everolimus PK level, unless the duration of the change lasts more than two weeks. If this was to 
occur, and a PK sample needed to be collected, the sample should be true trough. A true trough level is ~24 
hours (+/- 4 hours) after the last dose was taken, and after 4 days of consistent dosing. 
 
If treatment is interrupted due to toxicity, study drug should not be resumed until recovery to grade  1 is 
achieved. If recovery takes 6 weeks or more, the patient should be discontinued from the study. If recovery 
takes less than 6 weeks, study drug can be reintroduced at the initial dose or a lower dose level depending on 
the toxicity type and grade (see Tables 3 and 4). These changes must be recorded on the Dosage 
Administration Record and Dose Adjustment Log CRFs, as appropriate.  
 

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Dose adjustments (reduction, interruption, or possible dose re-escalation to starting dose) may also occur based 
on safety findings. All doses taken by the patient and all dose changes during the study must be recorded on the 
CRFs. Dose adjustments will also be made in the placebo group according to the placebo adjustment protocol.  
 
7.7.4 Concomitant Medications 
 
Patients must be instructed not to take any additional medications (over-the-counter, herbal, or other products) 
during the study without prior consultation with the investigator. All medications taken within 30 days of starting 
study treatment should be reported on the Concomitant Medications/Significant Non-Drug Therapies CRF. The 
following guidelines must be adhered to during the study: 
 
• Investigational or commercial anti-proliferative agents other than study drug (including other mTOR inhibitors, 
e.g., sirolimus, temsirolimus) are prohibited. 
 
• Co-administration with moderate or strong inhibitors of CYP3A4 (e.g., ketoconazole, itraconazole, ritonavir, 
erythromycin, fluconazole) or inhibitors of P-glycoprotein (PgP) must be avoided (refer to Table 9 and Table 10) 
 
• Co-administration with strong inducers of CYP3A4, other than antiepileptics, must be avoided. Table 9 lists 
clinically relevant CYP3A inhibitors, inducers and the definition of strong and moderate inhibitors/inducers. 
 
• Seville orange, star fruit, grapefruit and their juices affect P450 and PgP activity. Concomitant use should be 
avoided 
 
Everolimus may affect the response to vaccinations making the response to the vaccination less effective.   As 
everolimus is an immunosuppressant, live vaccines should be avoided while a patient is treated with everolimus.  
 
Otherwise, the use of other concomitant medication/therapy deemed necessary for the care of the patient is 
allowed. The investigator should instruct the patient to notify the study site about any new medications he/she 
takes after the start of the study drug. All medications (other than study drug) and significant non-drug therapies 
(including physical therapy and blood transfusions) administered after the patient starts everolimus and for up to 
84 days (12 weeks) after study drug discontinuation must be listed on the CRF. 
 
A letter will be provided to each randomized patient’s primary care physician outlining medications to avoid. 
 
Table 9 Clinical relevant drug interaction: inducers and inhibitors of isoenzyme CYP3A 
I N D U CE R S 
Strong inducers: 
avasimibe, carbamazepine, enzalutamide, mitotane, phenobarbital, phenytoin, rifabutin, rifampin (rifampicin), St. 
John's wort (Hypericum perforatum) 
 
Moderate inducers: 
bosentan, efavirenz, etravirine, genistein, lersivirine, lopinavir, modafinil, nafcillin, ritonavir, semagacestat, 
talviraline, thioridazine, tipranavir 
 
Weak inducers: 
amprenavir, aprepitant, armodafinil (R-modafinil), bexarotene, boceprevir, brivacetam, clobazam, danshen, 
dexamethasone, Echinacea, eslicarbazepine, garlic (Allium sativum), gingko (Ginkgo biloba), ginseng, 
glycyrrhizin, methylprednisolone, nevirapine, oxcarbazepine, pioglitazone, prednisone, pleconaril, primidone, 
quercetin, raltegravir, ritonavir, rufinamide, sorafenib, Stribild (combo of elvitegravir, cobicistat, emtricitabine, a 
tenofovir), sulfinpyrazone,telaprevir, terbinafine, ticagleror, ticlopidine, topiramate, troglitazone, vemurafenib, 
vicriviroc/ritonavir, vinblastine  
 
I N H I B I T O R S 

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Strong inhibitors: 
boceprevir, 
clarithromycin, 
cobicistat, 
conivaptan, 
danoprevir/ritonavir, 
eltegravir/ritonavir, 
indinavir, 
indinavir/ritonavir, 
itraconazole, 
ketoconazole, 
lopinavir/ritonavir, 
mibefradil, 
nefazodone, 
nelfinavir, 
posaconazole, 
ritonavir, 
saquinavir, 
saquinavir/ritonavir, 
telaprevir, 
telithromycin, 
tipranavir/ritonavir, 
troleandamycin, voriconazole 
 
Moderate inhibitors: 
Amprenavir, aprepitant, atazanavir, atazanavir/ritonavir, casopitant, cimetidine, ciprofloxacin, crizotinib, 
cyclosporine, darunavir, darunavir/ritonavir, diltiazem, dronedarone, erythromycin, fluconazole, fosamprenavir, 
grapefruit juice (Citrus parasidi fruit juice), imatinib, lomitapide, netupitant, nilotinib, Schisandra sphenanthera, 
tofisopam, verapamil 
 
Weak inhibitors: 
almorexant, alprazolam, alprazolam, amiodarone, amlodipine, amlodipine, atorvastatin, azithromycin, 
berberine, bicalutamide, bicalutamide, blueberry juice, cilostazol, cilostazol, cimetidine, clotrimazole, 
clozoxazone, cranberry juice, cyclosporine, delavirdine, everolimus, fluoxetine, fluvoxamine, fosaprepritant, 
ginkgo, goldenseal, isoniazid, isoniazid, ivacaftor, lacipidine, linagliptin, nilotinib, oral contraceptives, 
pazopanib, peppermint oil, propiverine, ranitidine, ranitidine, ranolaxine, ranolazine, resveratrol, roxithromycin, 
Seville orange, simeprevir, sitaxentan, tabimorelin, tacrolimus, teriflunomide, ticagrelor, tipranavir/ritonavir, 
tolvaptan, zileuton 
 
Table 10 Clinical relevant drug interactions medicated by PgP 
PgP Substrates 
PgP Inhibitors  
PgP Inducers 
afatinib, alfuzosin, aliskiren, alogliptin, ambrisentan, 
apixaban, apremilast, aprepitant, atorvastatin acid, 
atorvastatin, azithromycin, boceprevir, bosentan, 
carvedilol, caspofungin, ceritinib, cerivastatin, 
citalopram, colchicine, CP-481,715, cyclosporine, 
dabigatran, digoxin, docetaxel, domperidone, 
doxepin, doxorubicin, eribulin, everolimus, fentanyl, 
fexofenadine, fidaxomicin, fluvastatin, fosamprenavir, 
atifloxacin, idelalisib, iloperidone, indacaterol, 
indinavir, irbesartan, lacosamide, lapatinib, 
levetiracetam, levofloxacin, linagliptin, linezolid, 
loperamide, losartan, maraviroc, mirabegron, 
moxifloxacin, naloxegol, nateglinide, nevirapine, 
nintedanib, lodaterol, paclitaxel, pantoprazole, 
paroxetine, pazopanib, phenytoin, posaconazole, 
pravastatin, proguanil, quinidine, quinine, ranolazine, 
riociguat, risperidone, ritonavir, rivaroxaban, 
saquinavir, silodosin, simeprevir, simvastatin, 
sirolimus, sitagliptin, sofosbuvir, sorafenib, 
tacrolimus, telaprevir, tenofovir, ticagrelor, tipranavir, 
tolvaptan, topotecan, umeclidinium, valsartan, 
vardenafil, vincristine, voclosporin, voriconazole 
alogliptin, canaglifozin, 
cremophor RH40, curcumin, 
ketoconazole, lapatinib, 
lopinavir/ritonavir, mirabegron, 
propafenone, simepravir, 
valspodar, vandetanib, 
voclosporin 
avasimibe, 
carbamazepine, 
efavirenz, genistein, 
phenytoin, 
quercetin, rifampin, 
St John’s wort 
Reference: Internal Clinical Pharmacology Drug-drug interaction (DDI) memo, updated April-2015 which 
summarizes DDI data from three sources including the FDA’s “Guidance for Industry, Drug Interaction Studies,” 
the University of Washington’s Drug Interaction Database, and Indiana University School of Medicine's Drug 
Interaction Table.  
 
7.7.5 Concomitant Medication Dose Adjustments 
 
Samples for trough everolimus PK levels (drawn 0 to 2 hours before the next scheduled dose) will be taken two 
weeks (+/-  one week) after the following events: 
1. everolimus dose increase to a higher level than previously taken 
2. reduction in the dose of a CYP3A4 or pGP inducer (e.g. reduction in anticonvulsant dose) 
3. starting, or increasing the dose of, a CYP3A4 or pGP inhibitor 
Trough is not required for dose re-escalation to a previously-used dose; or for dose decreases. 
 

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7.7.6 Study Drug Interruption or Discontinuation 
 
An intent to treat approach will be used. All data acquired prior to termination for the reasons outlined below will 
be included in the primary analysis unless patient withdraws consent. Every effort will be made to conduct a final 
study visit with the participant and participants will be followed clinically until, if applicable, all adverse events 
resolve. 
 
The term “interruption” refers to a patient stopping the study medication during the course of the study, but then 
re-starting it at a later time in the study (as determined by their study doctor). 
 
The term “discontinuation” refers to a patient’s premature and permanent withdrawal from the study treatment. 
The reason for discontinuation from treatment will be recorded. The patient may discontinue participation in the 
study for any of the following reasons: 
1. adverse event(s) 
2. abnormal laboratory value(s) 
3. abnormal test procedure result(s) 
4. protocol deviation 
5. subject withdrew consent 
6. Withdrawal by the investigator 
7. lost to follow-up 
8. administrative problems 
9. intercurrent illness or event that precludes further visits to the study site or ability to evaluate disease 
(e.g.-mental status change, large pleural effusion).  
10. death 
 
If a patient has discontinued the study drug due to an unacceptable adverse event or an abnormal laboratory 
value, he/she should not have withdrawal of consent recorded as the reason for discontinuation. Instead, the 
reason for discontinuation must be recorded as due to adverse event or abnormal laboratory value. 
 
All patients must have evaluations for 28 days after the last dose of study treatment. Participants that have 
ongoing adverse events at the 28 day follow-up visit will continue to be followed for an additional 8 weeks (56 
days) to record any SAEs that are suspected to be related to study drug. Patients lost to follow up should be 
recorded as such on the CRF. Patients who discontinue study drug before completing the study should be 
scheduled for a visit as soon as possible, at which time all of the assessments listed for End of Treatment visit 
will be performed. At a minimum, all patients who discontinue study treatment, including those who refuse to 
return for a final visit, will be contacted for safety evaluations during the 28 days following the last dose of study 
drug. Patients will also be asked a series of neurocognition follow-up questions by phone and will complete the 
CBCL and DCDQ at the final follow-up call.  
 
7.7.7 Withdrawal from the study and study evaluation completion 
 
Patients may voluntarily withdraw from the study or be dropped from it at the discretion of the investigator at any 
time.  
 
As a general rule, if a patient discontinues study drug and later is withdrawn from the study, the reasons for 
study evaluation completion may include the following: 
 
Protocol deviation 
 
Subject withdrew consent 
 
Lost to follow-up 
 
Death 
 
Use of non-study systemic anti-PTEN therapy 
 
Administrative problems 
 
For patients who are lost to follow-up, the investigator should show due diligence by documenting in the source 
documents steps taken to contact the patient, e.g., dates of telephone calls, registered letters, etc.  
 
7.7.8 Study Discontinuation 

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The National Institutes of Health (NIH), Data and Safety Monitoring Committee (DSMC) and local IRBs (at their 
local site) have the authority to stop or suspend this trial at any time. This study may be suspended or closed if: 
 
Early stopping rules have been met  
 
Accrual has been met 
 
The study objectives have been met 
 
The Study Chair / Study Investigators believe it is not safe for the study to continue 
 
The DSMC suspends or closes the trial 
 
The NIH suspends or closes the trial 
 
The FDA suspends or closes the trial 
 
7.8 Good Clinical Practice and Human Subjects Training 
 
All personnel conducting human research as part of the Consortium will have completed an approved training 
course on human subjects’ protection as required by their local institution; training is updated, and will be current 
at the time they are engaged in Consortium clinical activities. All personnel are required to read and agree to 
adhere to the FDA Good Clinical Practice Guidelines. Documentation of trainings will be maintained by each site 
in their regulatory binder.  
 
7.9 Neuropsychological Evaluations 
 
The measures included were selected to cover the full age range included in the trial. This combination of age 
and developmental level does mean that floor or ceiling effects may be seen in a small proportion of study 
participants on some of the measures. The approach in the study will be to use very experienced testers to 
administer measures. The testers will be asked to attempt all direct measures on all participants, with the 
acknowledgement that, in some instances it may not be possible to complete all tasks. All measures will be re-
administered as per study protocols, regardless of previous floor or ceiling effects.  
 
7.9.1 Training and Reliability of Independent Evaluations (IEs) 
 
At each site, an experienced team will conduct study procedures guided by an operations manual. IEs, who are 
blind to treatment condition, will rate 3 measures in this study: CGI-S, CGI-I, and ADOS-2. Similar to previous 
studies, IEs will be trained to reliability criterion on the CGI-S, CGI-I, and ADOS-2 on 3 to 4 vignettes at the start 
and yearly after that 42,43_ENREF_16. If IEs fail to meet reliability criterion, they will be re-trained and given a 
second set of 3 vignettes for scoring. This procedure will be repeated as necessary to ensure that each rater 
has demonstrated reliability.  
 
7.9.2 Research Report 
 
Upon request, participants/parents will be provided with a research report with a summary of assessment 
scores. This report will be provided at the end of treatment.  
 
7.9.3 Main Efficacy Outcomes 
 
Neurocognitive Composite 
A composite neurocognitive score will be generated based on performance on objective measures. The 
composite index score will be computed in two ways. The first will be an average of measures evaluating 
working memory (SB-5 working memory subscale), processing speed (CPT-3 mean reaction time), and fine 
motor skills (Purdue Pegboard Test-average of both hands). The second will include the above average 
weighted by two-thirds and an average of the remaining standardized, norm-referenced neurocognitive 
measures (e.g., non-verbal ability, visuomotor skills, verbal learning, receptive and expressive language) 
weighted by one-third. The first method will be preferred if nearly complete data is observed on the 3 primary 
constructs (working memory, processing speed, and fine motor skills) in randomized patients. The second will 
be preferred if missing data is present on >20% of randomized patients. In order for any patient to be included in 
the study they must complete measures from at least one of the three primary efficacy constructs. If patients 
cannot complete the full Stanford Binet, the Mullen Scales of Early Learning may be substituted. In a similar 

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fashion, if the CPT-3 cannot be completed, the processing speed index of the Wechsler scales may be 
substituted as an alternative measure of processing speed. 
 
Measures were chosen based on previous empirical findings in a cross-sectional cohort study of PTEN patients 
with ASD. In this study, measures of working memory and processing speed were differentially impaired relative 
to other cognitive measures, including IQ. Furthermore, the cognitive deficits seen in these patients were related 
to reduced PTEN protein levels and brain white matter abnormalities. Specifically, in a cross-sectional 
mediational model, reduced PTEN protein levels led to greater brain white matter abnormalities which, in turn, 
led to greater cognitive difficulties. Although not evaluated using standardized testing, in our prior cohort of 
PTEN patients with ASD, every patient had a history of fine motor difficulties, occupational therapy, and/or 
observations during testing of significant fine motor weaknesses. Motor difficulties are also consistent with brain 
white matter abnormalities. Thus, the neurocognitive index will include measures that have both empirical and 
biological bases for inclusion as outcome measures. 
 
In the second version of the neurocognitive composite, the primary justifications for including the remaining 
neurocognitive measures are: 1) if significant missing data is seen on measure of processing speed, working 
memory, and fine motor skills, including a minor component of additional neurocognitive measures will ensure 
more reliably obtained data is contributing to the measurement of change, and 2) PTEN expresses in all of the 
cells of the body, the brains of PTEN patients showed widespread changes that included white matter 
abnormalities but also regional gray matter changes, and mouse models of PTEN loss have suggested that 
dendrite and synaptic dysfunction is also a consequence. Thus, it is reasonable to expect a range of 
neurocognitive functions might improve. Inclusion of other neurocognitive measures as the minority part of the 
index will ensure that improvements in other domains, such as expressive language, also have an opportunity to 
contribute to individual patient outcome measurements. Given that we do not yet know with certainty which 
neurocognitive functions are most likely to improve in this whole brain condition, allowing for possible 
improvement in other domains seems prudent. 
 
Use of a neurocognitive composite score has several advantages. Assuming that everolimus treatment does not 
impair some functions while improving others (an unlikely scenario), a composite score is likely to be highly 
reliable, can be computed in all patients even if 1 or 2 measurements are missing or could not be completed, 
and has good domain coverage to capture possible variability across patients in the specific neurocognitive 
domains showing improvement. An example of the latter advantage, is if some individuals show improvements 
in processing speed while others show improvements in working memory. The neurocognitive composite would 
identify improvements in both of these sets of patients. As such, the neurocognitive composite may be more 
sensitive to change than any individual measure. Furthermore, a composite primary efficacy outcome will not 
require additional multiple comparison correction. 
 
7.9.4 Additional Secondary Efficacy Outcomes 
 
Attention Improvement 
Conners’ Continuous Performance Test, Third Edition (CPT-3/K-CPT-2): Discriminability index (d’) and 
Omissions  
 
Processing Speed Improvement 
Conners’ Continuous Performance Test, Third Edition (CPT-3/K-CPT-2): Mean Reaction Time 
Weschler Processing Speed Index Subtests: Processing speed if CPT cannot be administered 
 
Impulsivity Improvement 
Conners’ Continuous Performance Test, Third Edition (CPT-3/K-CPT-2): Commissions and Bias (B’) 
 
Working Memory Improvement 
Stanford-Binet Intelligence Scales, Fifth Edition (SB-5) Working Memory: Standard Score 
 
Global Clinical Improvement 
Clinical Global Improvement Scale; Severity and Improvement subscales 
 
Improving Global Cognitive Ability 

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Stanford-Binet Intelligence Scales, Fifth Edition (SB-5) or Mullen Scales of Early Learning; Full scale, verbal 
and nonverbal ability (IQ) 
 
Improving Long-Term Memory 
Wide Range Assessment of Memory and Learning, Second Edition (WRAML-2) – Verbal Learning: Scaled 
Score  
 
Improving Language 
Peabody Picture Vocabulary Test (PPVT-4): Standard Score 
Expressive Vocabulary Test Second Edition (EVT-2): Standard Score 
 
Improving Motor Deficits 
Purdue Pegboard (Pegs): Dominant and non-dominant hand combined standard scores 
Developmental Coordination Disorder Questionnaire (DCDQ): Total score 
 
Targeting Autism Symptoms 
Autism Diagnostic Observation Schedule (ADOS): Calibrated severity scores 
An interviewer-based, semi-structured observational schedule for the assessment of individuals with 
possible autism spectrum disorders. The ADOS is widely regarded as an excellent phenotyping and 
diagnostic aid, and is used in many large-scale autism studies throughout the world, including the 
AGRE (Autism Genetic Resource Exchange www.agre.org) and IMGSAC (International Molecular 
Genetics Study of Autism Consortium www.well.ox.ac.uk/monaco/autism/IMGSAC.shtml) studies.  
Autism Diagnostic Interview – Revised (ADI-R): Item scores (ever/most abnormal)/ current 
For children with mental age <18 months, clinical judgment should be used when determining the 
reliability/validity of an ADI-R administration. The ADI-R is a semi-structured diagnostic parent interview 
that probes for current behaviors and a developmental history consistent with autism symptomology. 
The ADI-R contains questions about an individual’s early development, communication, social 
interaction and patterns of behavior.  
Social Responsiveness Scale, Second Edition (SRS-2): Total T-score 
Repetitive Behavior Scale – Revised (RBS-R): Total raw score 
 
Improving Associated Behaviors 
Behavior Rating Inventory of Executive Function (BRIEF): General executive composite and sub-scale scores 
Adult/Child Behavior Checklist (CBCL ARF/CBCL): Total problems T score 
Short Sensory Profile (SSP): Total Score 
 
Improving Adaptive Behaviors 
Vineland Adaptive Behavior Scales-III (VABS-III) Caregiver report: Composite and scale scores. 
A measure of functional adaptive behavior, i.e. ability to cope with personal and social skills in everyday 
life that may be deficient in autism or developmental delays. Tester completes a questionnaire through 
interview with parent or caregiver. Any age. No parallel forms. Outcome measures: raw scores 
converted to scaled scores, then a summary value for a number of domains.  
 
Table 11 Neuropsychological Assessments Timeline 
Instrument 
Information 
Source 
Screening 
(Visit 1) 
Baseline^ 
(Visit 2) 
3-month# 
(Visit 5) 
6-month# 
(Visit 8) 
Primary Efficacy Outcome Neurocognitive 
Composite 
Testing 
 
X 
X 
X 
Stanford-Binet Fifth Edition (SB-5) / Mullen 
Scales of Early Learning (MSEL) 
Testing 
X 
 
 
X 
Columbia-Suicide Severity Rating Scale 
Interview 
X 
 
X 
X 
Stanford-Binet Working Memory Subscale 
Only 
Testing 
 
X 
X 
 
Conners’ Continuous Performance Test 
(CPT-3/K-CPT-2) 
Testing 
X 
X 
X 
X 
Wechsler Processing Speed Index (PSI) 
Testing 
 
X 
X 
X 
Wide Range Assessment of Memory and 
Testing 
 
X 
X 
X 

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Learning-2 (WRAML-2) 
Peabody Picture Vocabulary Test – Fourth 
Edition (PPVT-4) 
Testing 
 
X 
X 
X 
Expressive Vocabulary Test – Second 
Edition (EVT-2) 
Testing 
 
X 
X 
X 
Purdue Pegboard (PP) 
Testing 
X 
X 
X 
X 
Autism Diagnostic Observation Schedule 
(ADOS-2) 
Observational 
 
X 
 
X 
Social Responsiveness Scale – Second 
Edition (SRS-2) 
Parent/Caregiv
er/Informant or 
Self- Report 
 
X 
X 
X 
Repetitive Behavior Scale – Revised (RBS-
R) 
Parent/Caregiv
er/Informant or 
Self Report 
 
X 
X 
X 
Developmental Coordination Disorder 
Questionnaire (DCDQ) 
Parent/Caregiv
er/Informant or 
Self Report 
 
X 
X 
X 
Behavior Rating Inventory of Executive 
Function (BRIEF) 
Parent/Caregiv
er/Informant or 
Self  Report 
 
X 
X 
X 
Adult/Child Behavior Checklist (CBCL 
ARF/CBCL) 
Parent/Caregiv
er/Informant or 
Self  Report 
 
X 
X 
X 
Short Sensory Profile (SSP) 
Parent/Caregiv
er/Informant or 
Self  Report 
 
X 
X 
X 
Vineland Adaptive Behavior Scales (VABS-
III) – caregiver report 
 
Parent/Caregiv
er/Informant or 
Self  Report 
 
X 
X 
X 
Autism Diagnostic Interview (ADI-R) 
Direct Interview 
with 
Parent/Caregiv
er/Informant 
 
X 
 
 
Note: Individuals who are participating in the longitudinal study: Natural History of Individuals with Autism and Germline 
Heterozygous PTEN Mutations can leverage measures completed within +/- 6 month for this study at the discretion of the 
study psychologist. 
^ Procedures completed at the Double-Blind Month 6 visit will qualify as Open-Label baseline procedures, as applicable. 
# Visits occur in both Double-Blind and Open-Label treatment phases, as applicable. 
 
7.10 Dermoscopy Assessment 
 
A dermoscopy exam will occur at baseline, 3-month and 6-month visits. 
 
The dermoscopy assessment will be both non-invasive and objective. Exam will focus on the change in number, 
location, and size of skin manifestations commonly associated with PTEN mutations and will be performed by a 
physician at each site.  
 
If feasible, de-identified photos of skin lesions will be taken and later evaluated by a blinded independent rater. 
The independent rater will evaluate change in skin lesions over time with respect to size, shape, etc.  
 
7.11 Eye Tracking (Optional) 
 
If parents/patients opt-in to eye tracking tasks, they will be conducted at baseline, 3-month and 6-month visits.  
 
Eye tracking hardware. Gaze data will be collected using a remote eye tracker system available at all sites. 
Binocular gaze, 3D eye position, pupil, and timestamp data will be collected at a sampling rate of 60/120/250Hz. 
Gaze capture is automatically calibrated and provides position accuracy to 0.4° at a 60cm viewing distance 

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(spatial resolution 0.3°). Dwell time (proportion on target relative to total time on screen), time-to-first-fixation, 
and fixations (30 pixel area for 100ms) to regions-of-interest (ROIs) will be the primary measurements. 
Saccades between targets, revisiting between targets, and blink metrics will also be evaluated for potential 
validity. 
 
Remote eye gaze acquisition and scoring. Eye gaze data will be captured during viewing of a brief battery (7 
min) that consists of initial calibration and multiple stimuli from each of the following paradigms: dynamic 
individual faces, static side-by-side faces, joint attention bids, gaze following, reciprocal interactions, dynamic 
social versus dynamic geometric images, and passive viewing of social/object arrays. In each of the socially-
focused stimuli, elements will be coded as social or non-social ROIs. Dwell times/fixations will be computed to 
each a priori target ROI. Separate social and non-social attention indices will be computed by averaging dwell 
times to social and non-social targets, respectively. An aggregate attention index (see preliminary data) will be 
computed by averaging dwell times to social and non-social targets.  
 
Receptive language stimuli will also be shown at the end of the battery. These will include picture arrays with 
progressively larger numbers of stimuli (2, 4, 6, 8, 10 items per array) and more challenging stimuli (easy item: 
dog, hard item: tornado).44,45 Pictures will include common objects or actions. Time-to-first fixation to the 
receptive language target within 2 seconds of the end of the directive to look at the target (e.g., “Look at the 
ball”) will be computed. These times will be averaged across all receptive language stimuli to generate an 
average receptive language score.  
 
For the whole battery, only individuals showing at least 50% total time on screen during the viewing period will 
be included in analyses, based on recent recommendations for eye tracking in children with autism.46 For both 
the attention indices and receptive language score, fixation times will be manually inspected to detect outliers 
and distributions will be transformed, if needed. All stimuli will be presented using a standard package. 
Fixation/dwell times and time-to-first-fixation will be computed using available software systems. Data from the 3 
sites will be transferred to Cleveland Clinic where it will be analyzed under the consultation of Thomas Frazier, 
PhD. 
 
7.12 Resting State Encephalography and Auditory Evoked Potential (Optional) 
 
Resting state encephalography (EEG) and auditory evoked potential (AEP) procedures are optional. Resting 
state encephalography (EEG) and auditory evoked potential (AEP) data collection will occur at baseline, 3-
month, and 6-month visits if the parents/patients have opted in on the consent form. 
 
To our knowledge, there have been no prior studies of quantitative EEG or AEP in individuals with PTEN 
mutations.  Therefore, a key goal of including the EEG and AEP components in this trial is to offer the 
opportunity to collect and analyze such data.  Given this knowledge gap, the choice of paradigms and proposed 
analyses were informed by (a) EEG findings in a mouse model of PTEN that offer translational potential, and (b) 
EEG and AEP findings in individuals with developmental synaptopathies and/or symptomatology related to 
PTEN. 
 
In PTEN conditional knockout mice, loss of PTEN in cortical GABAergic neurons leads to a loss of 
somatostatin+ interneurons, an increased ratio of parvalbumin/somatostatin interneurons, and thus increased 
inhibition of glutamatergic cortical neurons.  In these mice, baseline prefrontal gamma power (particularly in the 
high gamma range, 62-90Hz) is reduced compared to that of wild type mice, but is significantly increased 
(compared to wild type) during a social interaction task.47  It is also worth noting, given the increased risk of 
autism in individuals with PTEN mutations, that individuals with autism spectrum disorder are often found to 
have  increases in baseline gamma power on EEG.48  Additionally, increased baseline gamma power has been 
seen in other genetic disorders on the P13K/AKT/mTOR and MAPK pathways, including tuberous sclerosis,49 
Fragile X,50 and 16p11.2 deletions (preliminary data). 
 
Altered temporal coordination of neural oscillations, as measured by altered intra-individual variability  and 
altered habituation in responses to a repetitive stimuli, has also been seen in a number of disorders in which 
symptomatology overlaps with that frequently seen in individuals with PTEN mutations.51 For example, in 
individuals with Fragile X, an auditory gating task reveals decreased habituation of the N1 event-related 
potential response between the first and second tone in a train of repetitive tones.  Such altered gating has been 
linked to altered sensory processing,52 and to behaviors associated with autism.53 Additionally, individuals with 

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Fragile X and idiopathic autism demonstrate altered phase locking, a measure of intra-individual response 
variability to repeated stimuli.54,55   Of note, it has been suggested that such findings may be related to the 
activity of parvalbumin+ and somatostatin+ interneurons.54,56,57  
 
Taken together, these findings lead to a hypothesis that the altered relationship between excitation and 
inhibition seen in PTEN conditional knockout mice may result in abnormalities in baseline gamma power on 
EEG and altered response variability and habituation on an auditory gating paradigm.  In turn, abnormal EEG 
and AEP activity may be associated with some of the neurobehavioral findings frequently seen in PTEN.   The 
paradigms chosen in this study were chosen primarily for the likelihood that individuals with PTEN mutations 
may demonstrate abnormalities on these paradigms, and thus for the potential that in the future such paradigms 
could be used as a biomarker to measure change over time in response to treatment.  In addition, while this 
particular study does not include a control group without PTEN mutations, the paradigms were chosen for their 
overlap with other ongoing studies, to potentially allow for comparisons of findings in the future.  For example, 
the baseline EEG paradigm is currently being used for several other studies examining EEG findings in 
individuals with idiopathic autism, genetic disorders such as TSC, and typically developing controls.  The AEP 
paradigm, in addition to having been used in Fragile X, is currently being used in a study of individuals with 
Phelan-McDermid syndrome, which is also part of the Developmental Synaptopathies Consortium. 
 
Resting EEG Procedures: Resting state EEG has been used increasingly to quantitatively characterize and 
track outcomes in a range of neuropsychiatric populations, including idiopathic autism. EEG leads will be placed 
on the participant’s head, and EEG data will be collected while participants are presented with non-social, 
abstract moving images in random order.  The resting EEG paradigm will take about 5 minutes. 
 
AEP Procedures: Auditory evoked potentials will be collected using the same EEG system as the resting EEG, 
with clicks as the auditory input. Subjects will passively listen to 150 sets of two 5 ms broadband noise bursts 
(65 db) separated by an inter-stimulus interval of 500 ms with inter-set intervals of 4,000 ms (total duration=~12 
mins).  Offline, data will be average referenced and filtered for time-frequency analyses. 
 
EEG/AEP data from the three sites will be transferred and analyzed by April Levin, M.D.   The data will be 
transferred from CCF and Stanford to Boston by using Children’s Hospital Boston File Transfer (CHBFT). This 
system has been approved by the IRB for use of secure file transferring. Once transferred, data will be stored at 
BCH on a password-encrypted computer and only research staff will have access to the files. 
 
 
Along with the EEG/AEP data, photographs will be taken of the electrode placements on participants’ head. 
These photos will allow the research staff to view electrode placement and make informed decisions concerning 
poor data in relation to incorrect electrode placement. Participants will also be videotaped during the EEG and 
AEP procedures. By videotaping participants, researchers will be able to determine whether abnormal EEG data 
may be due to the participant’s behavior. The photographs and videos will allow the research staff to ensure 
appropriate collection methods are used. Both the photographs and the videos that are collected will be sent to 
Dr. April Levin at Boston Children’s Hospital via secure methods. This data will be stored on securely at the 
DMCC. 
 
7.13 Safety Outcomes 
 
The Dosage Record and Treatment Emergent Symptom Scale (DOTES): This is a general rating scale 
published by the Early Clinical Drug Evaluation Unit of the National Institute of Mental Health. The scale has 
been widely used clinically for both children and adults to assess many central nervous system side effects as 
well as some behavioral side effects. It will be completed at screening, baseline, and at each subsequent visit. 
In addition, patients will be interviewed using the C-SSRS to proactively assess patients for suicidality and mood 
disturbances. If the patient’s and/or parents’ responses are of concern, the investigator performing the interview 
may refer them to a mental health professional at the hospital. More detailed information about the C-SSRS can 
be found in section 4.6.5. Specific adverse events will also be classified and graded according to Common 
Terminology Criteria for Adverse Events (CTCAE) v5.0.  Use of CTCAE will allow direct comparison with past 
and current clinical trials with everolimus, including the neurocognitive trial described in previous sections 
(NCT01289912). Vital signs (blood pressure, pulse, and temperature), height and weight will be obtained during 
screening, baseline, and at each subsequent visit.  
 
Table 12. Primary and Secondary Efficacy and Safety Outcome Measures. 

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Domain 
Measure 
 
Primary 
Efficacy 
Outcome 
Neurocognitive 
Composite 
Average working memory (SB-5 working memory), processing speed (CPT 
mean reaction time) and fine motor (Purdue Pegboard-average of both 
hands) subtests weighted at 2/3 of the composite score and the average of 
all other available neurocognitive testing measures (receptive and 
expressive language, non-verbal ability, verbal learning, sustained attention, 
impulsivity, and visuomotor skills) weighted 1/3 of the score.  
 
Secondary 
Efficacy 
Outcomes 
Global Ability 
Stanford-Binet-5 (SB-5): Full scale IQ, Verbal IQ, and Non-Verbal IQ or the 
Mullen Scales of Learning: Cognitive IQ 
Attention 
Conners’ Continuous Performance Test -3 (CPT-3/K-CPT-2): 
Discriminability (d’) and Omissions 
Processing Speed 
Conners’ Continuous Performance Test -3 (CPT-3/K-CPT-2): Mean 
Reaction Time; in cases where an individual cannot complete the CPT we 
will administer the appropriate Wechsler processing speed index subtest.  
Impulsivity 
Conners’ Continuous Performance Test (CPT): Commissions and Bias 
Long-Term Memory 
WRAML-2 Verbal Learning: Scaled score 
Language 
Peabody Picture Vocabulary Test -4 (PPVT-4): Standard Score 
Expressive Vocabulary Test – 2 (EVT-2): Standard Score 
 
Motor Functioning 
Purdue Pegboard (Pegs): Dominant and non-dominant hand standard 
scores 
Motor Coordination 
Developmental Coordination Disorder Questionnaire (DCDQ): Total score 
Autism Symptoms 
Autism Diagnostic Observation Schedule (ADOS): Calibrated severity score 
Social Responsiveness Scale – 2 (SRS-2): Total T-score 
Repetitive Behavior Scale – Revised (RBS-R): Total raw score 
Other Behavioral 
Symptoms 
Behavior Rating Inventory of Executive Function (BRIEF) - Global Executive 
Composite: Standard Score 
Child Behavior Checklist - Total Problems: Standard Score 
Sensory 
Processing 
Short Sensory Profile (SSP): Total Score 
Adaptive Behavior 
Vineland Adaptive Behavior Scales (VABS-III): Composite Standard Score 
Global Severity and 
Improvement 
Clinical Global Impressions-Severity (CGI-S) 
Clinical Global Impressions-Improvement (CGI-I) 
 
7.14 Vital Signs 
 
Pulse, respiration rate, blood pressure and temperature, height and weight will be measured as indicated in the 
Assessment schedule (Table 13).  Blood pressure, pulse and respiration rate should be measured on patients 
after at least 3 minutes in the sitting position.  
 
7.15 Body Surface Area 
 
Body Surface Area (BSA) should be calculated before the baseline visit. The measurements taken at the 
screening visit should be used to perform this calculation. The same process will be used at the Month 1 visit 
(i.e. using the baseline height and weight for the calculation) to ensure the patient’s dose has not changed.  BSA 
(in 
m2) 
should 
be 
calculated 
via 
the 
Cornell 
online 
BSA 
calculator: 
http://www.users.med.cornell.edu/~spon/picu/calc/bsacalc.htm, or by using the following formula where weight 
(W) is in kilograms and height (H) is in centimeters (Dubois and Dubois, 1916):  
 
BSA = (W0.425x H0.725) x 0.007184 
 
The BSA will indicate the starting medication dose for all participants, which will be calculated for the baseline 
and Month 1 visits. After the Month 1 visit, dosing should be based on the dose adjustments determined by the 
unblinded physician, and/or the study physician, as described in Section 6.2. The 2.5mg tablets will be used to 
make the adjustments. Sites may substitute 5mg or 10mg tablets as described in Section 6.2. 
 
7.16 Laboratory Evaluations 

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All standard clinical laboratory analyses described below are to be performed by the central laboratory 
(LabConnect and their subcontracted entities). PK samples and serology panels will be processed by ARUP 
Laboratories. Blood may be drawn at the site, physician office or by traveling phlebotomist as appropriate, 
according to the Visit Schedule outlined in Table 13. The volume of sample collection is listed in Table 14.  
 
7.16.1 Hepatitis and HIV Screening 
 
Patients who have a positive test for hepatitis B, hepatitis C, or HIV at screening must be excluded from the 
study unless the positive Hepatitis B antibody test resulted from prior vaccination. The hepatitis and HIV tests for 
antibodies will be done only at screening, at the principal investigator’s discretion, based off the risk factor 
assessment (see Table 13).   
 
7.16.2 Hematology  
 
Hematology tests are to be performed at each scheduled onsite visit (± 14 days) as indicated in  
Table 13. These must include: hemoglobin, hematocrit, platelets, red blood cell count (RBC), total white blood 
cell count (WBC) absolute & differential including neutrophils, lymphocytes, monocytes, eosinophils, basophils). 
Absolute neutrophil count (ANC) will be calculated by the laboratory.    
 
7.16.3 Coagulation 
 
Prothrombin time will be determined at screening, 3 months and 6 months (end of treatment) will be reported as 
international normalized ratio (INR).  In addition, fibrinogen and partial thromboplastin (PTT) will be determined 
at screening, 3 months and 6 months.  
 
7.16.4 Serum Pregnancy/Hormone Testing 
 
Serum Pregnancy 
All females of childbearing potential must have a negative serum pregnancy at screening. Urine pregnancy tests 
may be used for onsite visits (if deemed clinically necessary).  A serum pregnancy test will be administered 
again at the final six-month visit.  
 
Adequate contraception, excluding estrogen containing contraceptives, must be used on-study and for up to 8 
weeks after ending treatment.  Abstinence will be considered an acceptable contraceptive.  
 
To ensure patient safety, each pregnancy in a patient on study drug must be reported to Novartis within 24 
hours of learning of its occurrence. The pregnancy should be followed up to determine outcome, including 
spontaneous or voluntary termination, details of the birth, and the presence or absence of any birth defects, 
congenital abnormalities, or maternal and/or newborn complications. 
 
Pregnancy should be recorded on a Clinical Trial Pregnancy Form and reported by the investigator to the local 
Novartis Integrated Medical Safety (“IMI”) Department. Pregnancy follow-up should be recorded on the same 
form and should include an assessment of the possible relationship to the Novartis study drug of any pregnancy 
outcome. Any SAE experienced during pregnancy must be reported on the SAE Report Form. 
 
Male Contraception 
Sexually active males must use a condom during intercourse while taking the drug and for 8 weeks after 
stopping treatment and should not father a child during this period. 
 
Pregnancy outcomes must be collected for the female partners of any males who took study drug in this study. 
Consent to report information regarding these pregnancy outcomes should be obtained from the mother.  
 
Hormone Testing 
For all participants, female or male, over the age of 7 years, a hormone test will be done at the screening visit 
and again at the month 6 visit. 
 

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For all participants under the age of 7 years, a sample will be drawn for estradiol, LH, and FSH at the screening 
visit and again at the month 6 visit. 
 
The potential for everolimus to cause infertility in male and female patients is unknown. However, menstrual 
irregularities, secondary amenorrhea and associated luteinizing hormone (LH)/follicle stimulating hormone 
(FSH) imbalance has been observed. Based on non-clinical findings, male and female fertility may be 
compromised by treatment with everolimus.  
 
7.16.5 Biochemistry and Lipid Profile 
 
The following tests will be performed at each scheduled onsite visit (Screening, Month1, Month 3, Month 6 
visits) (± 14 days) and will include sodium, potassium, chloride, bicarbonate, creatinine, albumin, total protein, 
SGOT (AST), SGPT (ALT), total bilirubin, alkaline phosphatase, uric acid, BUN, calcium, magnesium, 
phosphate, total LDH, and fasting glucose. A sample will be drawn for T4 and TSH at the screening visit and 
again at the 6 month visit. 
 
In order to assess renal function during the trial, an age appropriate measure, either the 
Cockcroft-Gault (1976) formula the Schwartz (2009) formula.  
 
The Cockcroft-Gault formula, for patients 18 and older, is shown below, where creatinine clearance is “x” (in 
Ml/min), age is measured in years, weight in kg, creatinine in μmol/L, and the constant “K” is 1.23 for men and 
1.04 for women (Cockcroft-Gault, 1976). 
 
𝑥 = 
(140 −𝐴𝑔𝑒) 𝑥 𝑊𝑒𝑖𝑔ℎ𝑡 𝑥 𝐾
𝑆𝑒𝑟𝑢𝑚 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒
 
 
 
The Schwartz formula is shown below, where Glomerular Filtration Rate (GFR) is “x” (in 
Ml/min/1.73 m2), height is measured in cm and serum creatinine in mg/Dl (Schwartz et al, 2009). 
 
𝑥 = 
0.41 𝑥 𝐻𝑒𝑖𝑔ℎ𝑡
𝑆𝑒𝑟𝑢𝑚 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒
 
 
A lipid profile (cholesterol, triglycerides, LDL, HDL) will be determined at screening, 3 months and 6 months 
(end of treatment). The patient must be in a fasting state at the time of blood sampling for this evaluation. 
 
7.16.6 HbA1c 
 
A HbA1c, hemoglobin A1c, test will be collected from patients at the screening visit. This test monitors a 
patient’s average blood glucose over the last 2 to 3 months. The test will inform study staff about a patient’s 
fasting glucose and/or diabetes mellitus, which is used when determining eligibility. Uncontrolled diabetes 
mellitus as defined by HbA1c >8% despite adequate therapy, will be exclusionary. Patients with a known history 
of impaired fasting glucose or diabetes mellitus (DM) may be included, however blood glucose and antidiabetic 
treatment must be monitored closely throughout the trial and adjusted as necessary; 
 
 
7.16.7 Urinalysis 
 
A standard urinalysis assessment (pH, protein, glucose, blood, ketones, and leukocytes) should be performed at 
each onsite visit. This must be supplemented with laboratory quantification of any potentially relevant 
abnormalities when dipstick test is abnormal.   
 
7.16.8 Blood Sample for Everolimus (Everolimus Levels) 
 
A maximum volume of 3 ml of blood will be drawn for a trough everolimus level. Blood draws will be timed to 
occur at 24 ± 4 hours after ingestion of the drug. Everolimus levels will be measured at ARUP Laboratories 
associated with LabConnect in Salt Lake City, UT. For families that do not live close to the study site, there are 

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two options for remote blood collection. A kit for remote collection of blood samples can be sent to the 
participant and returned per the shipping instruction by express mail, or a designated and trained remote health 
care provider can collect a PK sample and ship it per the shipping instruction by express mail. A central 
unblinded investigator will be responsible for dose adjustment in both the treatment and placebo arms. If the 
everolimus level falls outside the range of 5-15 ng/ml, the local research team and PI will be notified that a dose 
adjustment is needed, the dose adjustment will be implemented by the local investigator per protocol and 
unblinded investigator direction, and the blood level will be redrawn two weeks (+/- one week) later. The site 
team is blinded with respect to treatment assignment at all times. This process will be repeated until the 
everolimus level falls within the target range. Dose adjustments will be randomly pre-assigned to occur in the 
placebo arm of this trial. The DMCC will assign dose adjustments to placebo randomization codes and share 
this information with the unblinded physician who will facilitate a dose adjustment at the appropriate time-point 
for these placebo participants. 
 
A pre-dose trough blood sample will be collected immediately prior to dosing at month 1 (visit 3).  This sample 
should be collected prior to the patient’s next dose of study drug ~24 ± 4 hours after the last dose. The central 
unblinded investigator will be responsible for dose adjustment, using the 2.5mg tablets, to achieve the target 
everolimus concentration range of 5-15 ng/ml. Sites may substitute 5mg or 10mg tablets as described in Section 
6.2. 
 
Additional pre-dose trough blood samples will be taken if a dose adjustment occurs due to toxicity or due to 
concomitant medication dose adjustment (See Section 7.6.3). Should clinically significant changes in the 
patient’s height and/or weight occur at any time point during the study, the principal investigator may choose to 
have additional pre-dose trough blood samples taken.  
 
Samples will be frozen as whole blood within 2 hours after collection at -80°C and transported to the central 
laboratory, LabConnect, for analysis. Everolimus concentrations will be analyzed in whole blood using a 
validated method.  
 
7.16.9 Additional Blood Sample for PTEN Associated Proteins 
 
An additional blood sample (3.5 ml) for future research studies will be allowed during the course of study 
participation with the subject’s consent and authorization. Samples will be obtained at three study time points 
(screening or baseline, 3-month visit, 6-month visit) for serum, RNA and plasma for future biomarker research 
projects regarding PTEN associated proteins. When possible, blood draw will be combined with clinically-
indicated venous blood tests to minimize the need for additional venipuncture and total blood volume for 
combined clinical and research studies will not exceed NIH recommended guidelines for subject age and 
weight. The consent form includes a checkbox for participants to decide if they want to provide an extra blood 
sample for future biomarker research projects. Samples will be de-identified and sent to the Genomic Medicine 
Institute Biorepository at Cleveland Clinic.  
 
7.16.10 Additional Blood Sample for Insulin Like Growth Factor Binding Protein 2 (IGFBP-2) 
 
An additional blood sample (3.5 ml) for IGFBP-2 analysis will be collected at the screening or baseline visit, 3 
month, and the 6 month visit. IGFBP is a useful biomarker of response, as over expression of IGFBP-2 has 
been associated with cancers and overgrowth syndromes. In a short treatment trial, we may be able to capture 
changes in IGFBP expression due to everolimus that shows promising evidence of therapeutic value in patients 
with PTEN. This measure will serve as a surrogate biomarker of effect. Samples will be de-identified and sent to 
the Genomic Medicine Institute Biorepository at Cleveland Clinic for analysis.  
 
7.16.11 Microbiome/Mycobiome Sample Collection  
 
When possible, we will collect optional saliva, urine, and fecal samples from all patients at baseline, month 3, 
and month 6 of the double-blind treatment phase and during the open label phase. Patients will receive the kits 
at home from Cleveland Clinic. Coordinators will instruct the participant/family to collect the samples ±14 days 
from the onsite visit. Once the samples are collected, the kit should be shipped back to Cleveland Clinic. The kit 
will also contain these instructions, all materials needed to collect the samples, and a return label. 
 

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These samples will be sent for storage at the Genomic Medicine Biorepository (GMB). If funding becomes 
available, future correlative studies will investigate PTEN pathway protein extraction and quantification, 
RNASeq, proteomic, microbiomic, and mycobiomic analyses. 
 
7.17 Data from Samples 
 
Data obtained from samples may also be shared with publically accessible databases, such as the database of 
Genotypes and Phenotypes (dbGaP). 
 
7.18 Medical Records 
 
We will collect medical records and clinical reports (if available) for screening and confirmation of medical 
history.  
 
All medications (other than study drug) and significant non-drug therapies (including physical therapy and blood 
transfusions) administered after the patient starts everolimus and for up to 56 days (8 weeks) after study drug 
discontinuation must be listed on the CRF.  All AEs and SAEs, regardless of causality will be collected and 
recorded up until 56 days (8 weeks) after study drug has been discontinued. This information will be collected 
on the CRFs listed under the phone call that happens after 1-month (28-days) of stopping study medication 
and/or completing the trial. 
 
7.19 Follow-Up Questionnaires 
 
All medications (other than study drug) and significant non-drug therapies (including physical therapy and blood 
transfusions) administered after the patient starts everolimus must be listed on the CRF.  All AEs and SAEs, 
regardless of causality will be collected and recorded until the study drug has been discontinued. This 
information will be collected by phone call at 28 days post-treatment termination and for 8 additional weeks 
after, as indicated.  
 
At the follow-up time point we will collect information about behavior (CBCL) and motor skills (DCDQ) as well as 
overall neurocognitive outcome. Parental questionnaires will be sent home and completed by the family of the 
participant. The questions about overall neurocognitive outcome will be conducted over the phone.  
 
 
 
Table 13A: Double-Blind Treatment Phase Study Timeline 
Measurement 
Performed 
by 
Rationale 
Scree
n 
(Visit 
1) 
Baseline2 
(Visit 2)  
Month 
1 
(Visit 3) 
Month 2* 
(Visit 4) 
Month 
3 
(Visit 5) 
Month 4* 
(Visit 6) 
Month 5* 
(Visit 7) 
Month 
6 
(Visit 8) 
Follow-
up 
 
 
PRN 
Visit Windows 
 
 
 
Within 
42d 
of 
Screen 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
28d after 
last visit 
+/-14d 
 
Medical History 
Clinician 
Eligibility 
X 
X 
X** 
X 
X** 
X 
X 
X** 
X 
 
Clinical Interview 
Clinician 
Eligibility + 
Safety 
X 
X 
X** 
X 
X** 
X 
X 
X** 
 
 
Inclusion/ 
Exclusion criteria 
Clinician 
Eligibility 
X 
X 
 
 
 
 
 
 
 
 
CGI Scale: 
Severity & 
Improvement 
IE 
Efficacy 
 
X3 
X** 
X5 
X** 
X5 
X5 
X** 
 
 
Vital Signs 
Clinician 
Safety 
X 
X 
X 
 
X 
 
 
X 
 
 
Physical / 
Neurological 
Exam 
Clinician 
Safety 
X 
 
 
 
 
 
 
X** 
 
X 
Dermoscopy 
Clinician 
Efficacy  
 
X 
 
 
X** 
 
 
X** 
 
 

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Microbiome/ 
Mycobiome 
Sample 
Collection 
 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 
Tanner Staging 
Clinician 
Safety 
 
X 
 
 
 
 
 
X 
 
 
Side-effects 
(CTCAE v5.0 + 
DOTES) 
Clinician 
Safety 
X 
X 
X** 
X 
X** 
X 
X 
X** 
X 
 
Laboratory tests1 
Lab 
Safety 
X 
 
X 
 
X 
 
 
X 
 
X 
Everolimus level 
(only done after 
M1 if needed) 
Lab 
Dose Adjust 
 
 
X 
 
 
 
 
 
 
X 
PTEN-associated 
proteins 
Lab 
Efficacy 
X4 
X4 
 
 
X 
 
 
X 
 
 
Concomitant 
treatment log 
Clinician 
Safety+ 
Efficacy 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Autism 
Diagnostic 
Interview- 
Revised 
Clinician 
Sample 
Description 
 
X 
 
 
 
 
 
 
 
 
Primary Efficacy 
Outcome 
Neurocognitive 
Composite 
Clinician 
Efficacy 
 
X 
 
 
X 
 
 
X 
X 
 
Columbia-
Suicide Severity 
Rating Scale 
Clinician 
Safety 
X 
 
X** 
 
X** 
 
 
X** 
 
 
Conners’ 
Continuous 
Performance 
Test (CPT-3/K-
CPT-2) 
Clinician 
Efficacy 
X 
X 
 
 
X 
 
 
X 
 
 
Stanford-Binet 
Fifth Edition (SB-
5) / Mullen 
Scales of Early 
Learning (MSEL) 
Clinician 
Efficacy 
X 
 
 
 
 
 
 
X 
 
 
Stanford-Binet 
Working Memory 
Subscale Only 
Clinician 
Efficacy 
 
X 
 
 
X 
 
 
 
 
 
Purdue 
Pegboard (PP) 
Clinician 
 
Efficacy 
X 
X 
 
 
X 
 
 
X 
 
 
Wechsler 
Processing 
Speed Index 
Clinician 
 
 
X 
 
 
X 
 
 
X 
 
 
Wide Range 
Assessment of 
Memory and 
Learning-2 
(WRAML-2) 
Clinician 
Efficacy 
 
X 
 
 
X** 
 
 
X** 
 
 
Peabody Picture 
Vocabulary Test 
– Fourth Edition 
(PPVT-4) 
Clinician 
Efficacy 
 
X 
 
 
X** 
 
 
X** 
 
 
Expressive 
Vocabulary Test 
– Second Edition 
(EVT-2) 
Clinician 
Efficacy 
 
X 
 
 
X** 
 
 
X** 
 
 
Autism 
Diagnostic 
Observation 
Schedule 
(ADOS-2) 
Clinician 
Efficacy 
 
X 
 
 
 
 
 
X 
 
 
Social 
Responsiveness 
Scale – Second 
Edition (SRS-2) 
Parent 
Report 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 

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Repetitive 
Behavior Scale – 
Revised (RBS-R) 
Parent 
Report 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 
Developmental 
Coordination 
Disorder 
Questionnaire 
(DCDQ) 
Parent 
Report 
Efficacy 
 
X 
 
 
X 
 
 
X 
X 
 
Behavior Rating 
Inventory of 
Executive 
Function (BRIEF) 
Parent 
Report 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 
Adult/Child 
Behavior 
Checklist 
Parent 
Report 
Efficacy 
 
X 
 
 
X 
 
 
X 
X 
 
Sensory Profile 
Questionnaire - 
Short Form 
(SPQ) 
Parent 
Report 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 
Vineland 
Adaptive 
Behavior Scales 
(VABS-III) – 
caregiver report 
Parent 
Report 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 
Eye Tracking 
(Optional) 
Clinician 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 
Resting State 
EEG/Auditory 
Evoked 
Potentials 
(Optional) 
Clinician 
Efficacy 
 
X 
 
 
X 
 
 
X 
 
 
Participant 
Unblinding 
 
 
 
 
 
 
 
 
 
X** 
 
X 
1Coagulation testing will only be performed at screening, month 3 and month 6. Pregnancy testing, serum is done at 
screening and month 6 and urine may be used for onsite visits (if deemed clinically necessary) for women of childbearing 
potential. 
2Monthly visit windows are calculated based on date of Baseline visit. 
3At the baseline visit, only the CGI: Severity scale will be completed. 
4PTEN-associated protein blood collections will be collected at either the screening or baseline visit (as well as at the 3 
month and 6 month visits) 
5CGI optional for phone interviews month 2, month 4, and month 5. 
 
CGI: Clinical Global Impressions Scale; IE: Independent Evaluator; CTCAE: Common Terminology Criteria for Adverse 
Events;  DOTES: Dosage Record and Treatment Emergent Symptom Scale;**: primary outcome mesaure only. 
*= May be performed by phone or in person 
**= May be performed by phone/videoconference under extenuating circumstances (note: Physical / Neurological Exam can 
not be completed in full remotely, but a modified version can be performed) 
 
Table 13B: Open Label Treatment Phase Study Timeline 
Measurement 
Performed 
by 
Rationale 
Baseline2 
(Visit 9)  
Month 1 
(Visit 
10) 
Month 2* 
(Visit 11) 
Month 3 
(Visit 
12) 
Month 4* 
(Visit 13) 
Month 5* 
(Visit 14) 
Month 6 
(Visit 
15) 
Follow-
up 
 
 
PRN 
Visit Windows 
 
 
Within 
2 
weeks 
of 
Phase 
A, 
Month 6 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
28d after 
last visit 
+/-14d 
 
Medical History 
Clinician 
Eligibility 
# 
X** 
X 
X** 
X 
X 
X** 
X 
 
Clinical Interview 
Clinician 
Eligibility + 
Safety 
# 
X** 
X 
X** 
X 
X 
X** 
 
 
Inclusion/ 
Exclusion criteria 
Clinician 
Eligibility 
X 
 
 
 
 
 
 
 
 
CGI Scale: 
Severity & 
Improvement 
IE 
Efficacy 
X3** 
X** 
X4 
X** 
X4 
X4 
X** 
 
 
Vital Signs 
Clinician 
Safety 
 
X 
 
X 
 
 
X 
 
 
Physical / 
Neurological 
Exam 
Clinician 
Safety 
 
 
 
 
 
 
X** 
 
X 

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Dermoscopy 
Clinician 
Efficacy  
 
 
 
X** 
 
 
X** 
 
 
Microbiome/ 
Mycobiome 
Sample 
Collection 
 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Tanner Staging 
Clinician 
Safety 
 
 
 
 
 
 
X 
 
 
Side-effects 
(CTCAE v5.0 + 
DOTES) 
Clinician 
Safety 
 
# 
X** 
X 
X** 
X 
X 
X** 
X 
 
Columbia-Suicide 
Severity Rating 
Scale 
Clinician 
Safety 
 
 
X** 
 
X** 
 
 
X** 
 
 
Laboratory tests1 
Lab 
Safety 
# 
X 
 
X 
 
 
X 
 
X 
Everolimus level 
(only done after 
M1 if needed) 
Lab 
Dose 
Adjust 
 
X 
 
 
 
 
 
 
X 
PTEN-associated 
proteins 
Lab 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Concomitant 
treatment log 
Clinician 
Safety+ 
Efficacy 
# 
X 
X 
X 
X 
X 
X 
X 
 
Primary Efficacy 
Outcome 
Neurocognitive 
Composite 
Clinician 
Efficacy 
# 
 
 
X 
 
 
X 
X 
 
Conners’ 
Continuous 
Performance 
Test (CPT-3/K-
CPT-2) 
Clinician 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Stanford-Binet 
Fifth Edition (SB-
5) / Mullen 
Scales of Early 
Learning (MSEL) 
Clinician 
Efficacy 
 
 
 
 
 
 
X 
 
 
Stanford-Binet 
Working Memory 
Subscale Only 
Clinician 
Efficacy 
 
 
 
X 
 
 
 
 
 
Purdue Pegboard 
(PP) 
Clinician 
 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Wechsler 
Processing 
Speed Index 
Clinician 
 
 
 
 
X 
 
 
X 
 
 
Wide Range 
Assessment of 
Memory and 
Learning-2 
(WRAML-2) 
Clinician 
Efficacy 
 
 
 
X** 
 
 
X** 
 
 
Peabody Picture 
Vocabulary Test 
– Fourth Edition 
(PPVT-4) 
Clinician 
Efficacy 
 
 
 
X** 
 
 
X** 
 
 
Expressive 
Vocabulary Test 
– Second Edition 
(EVT-2) 
Clinician 
Efficacy 
 
 
 
X** 
 
 
X** 
 
 
Autism 
Diagnostic 
Observation 
Schedule 
(ADOS-2) 
Clinician 
Efficacy 
 
 
 
 
 
 
X 
 
 
Social 
Responsiveness 
Scale – Second 
Edition (SRS-2) 
Parent 
Report 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Repetitive 
Behavior Scale – 
Revised (RBS-R) 
Parent 
Report 
Efficacy 
 
 
 
X 
 
 
X 
 
 

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Developmental 
Coordination 
Disorder 
Questionnaire 
(DCDQ) 
Parent 
Report 
Efficacy 
 
 
 
X 
 
 
X 
X 
 
Behavior Rating 
Inventory of 
Executive 
Function (BRIEF) 
Parent 
Report 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Adult/Child 
Behavior 
Checklist 
Parent 
Report 
Efficacy 
 
 
 
X 
 
 
X 
X 
 
Sensory Profile 
Questionnaire - 
Short Form 
(SPQ) 
Parent 
Report 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Vineland 
Adaptive 
Behavior Scales 
(VABS-III) – 
caregiver report 
Parent 
Report 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Eye Tracking 
(Optional) 
Clinician 
Efficacy 
 
 
 
X 
 
 
X 
 
 
Resting State 
EEG/Auditory 
Evoked 
Potentials 
(Optional) 
Clinician 
Efficacy 
 
 
 
X 
 
 
X 
 
 
 
1Coagulation testing will only be performed at screening, month 3 and month 6. Pregnancy testing, serum is done at 
screening and month 6 and urine may be used for onsite visits (if deemed clinically necessary) for women of childbearing 
potential. 
2Monthly visit windows are calculated based on date of Baseline visit. 
3At the baseline visit, only the CGI: Severity scale will be completed. 
4CGI optional for phone interviews month 2, month 4, and month 5. 
 
CGI: Clinical Global Impressions Scale; IE: Independent Evaluator; CTCAE: Common Terminology Criteria for Adverse 
Events;  DOTES: Dosage Record and Treatment Emergent Symptom Scale;**: primary outcome mesaure only. 
*= May be performed by phone or in person 
**= May be performed by phone/videoconference under extenuating circumstances (note: Physical / Neurological Exam can 
not be completed in full remotely, but a modified version can be performed)#=procedures may be carried over from the 
Double-Blind Month 6 visit, if within 2 weeks of the visit. 
 
 
 
 
 
 
 
 
Table 14: 
 Blood Collection Amounts in mL 
Visit Name 
Screen 
(Visit 1) 
Baseline2 ^‡ 
(Visit 2)  
Month 1‡ 
(Visit 3) 
Month 2*‡ 
(Visit 4)# 
Month 3‡ 
(Visit 5) 
Month 4*‡ 
(Visit 6)# 
Month 5‡ 
(Visit 7)# 
Month 6‡ 
(Visit 8) 
PRN‡ 
Time point 
 
Within 42d of 
Screen 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
+/-14d 
 
 
Serum/RNA/plasma 
storage (optional) 
3.5× 
3.5× 
 
 
3.5 
 
 
3.5 
 
 
IGFBP-2 (optional) 
3.5× 
3.5× 
 
 
3.5 
 
 
3.5 
 
 
Serology 
Panel: 
Hepatitis B and C, or 
HIV (as needed) 
9.0 
 
 
 
 
 
 
 
 

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Hormone 
Testing/ 
Serum Pregnancy (if 
needed)* 
5.0 
 
 
 
 
 
 
5.0 
 
 
Chemistry Panel ** 
5.0 
 
4.0 
 
4.0 
 
 
5.0 
 
 
Hematology 
3.0 
 
3.0 
 
3.0 
 
 
3.0 
 
HbA1c 
3.0 
 
 
 
 
 
 
 
 
 
Coagulation Studies 
PT/PTT/INR 
4.5 
 
 
 
4.5 
 
 
4.5 
 
 
Everolimus level*** 
 
 
3.0 
 
 
 
 
 
 
3.0 
 
Total 
Blood 
Drawn 
(mL) 
36.5 
7.0 
10 
 
18.5 
 
 
24.5 
 
* For patients over the age of 7, hormone testing collected on all participants only at screening. For participants under the 
age of 7, estradiol, LH, and FSH will be tested. Serum Pregnancy collected at screening and Month 6 for female participants 
of childbearing potential.  
** Fasting glucose will constitute 2.0 mL of this amount and be collected in a separate tube. 
***Additional everolimus levels may be drawn if a dose adjustment occurs due to toxicity or due to concomitant medication 
dose adjustment. 
# Visits may occur in person or by phone, no blood collected. 
^  Procedures completed at the Double-Blind Month 6 visit will qualify as Open-Label baseline procedures, as applicable. 
‡ Visits occur in both Double-Blind and Open-Label treatment phases, as applicable. 
× Optional blood samples can be collected at either the screening or baseline visit 
 
7.20 Data Collection 
 
All data outlined in schedule of events and in sections below will be collected on case report forms. 
 
Screening data will be collected on screen-fails. Patients who complete the informed consent process and do 
not meet all entry criteria and therefore who do not receive everolimus or placebo will be considered screen 
failures. Screen failures should be documented as ineligible in the database.  
 
All study data will be collected via systems created in collaboration with the RDCRN Data Management and 
Coordinating Center and will comply with all applicable guidelines regarding patient confidentiality and data 
integrity.  
 
7.21 Inclusion/Exclusion Criteria  
 
Information regarding eligibility criteria will be entered on the Inclusion/Exclusion CRF, for both the double-blind 
and open-label phases.  Patients who do not meet all entry criteria for a given phase of the study should not be 
entered into that phase.   
 
7.22 Patient Demographics/Clinical Interview (Medical History) 
 
Data will be collected on patient characteristics including demographic information (age, sex, race, weight) and 
other background or relevant medical history (disease history, family history of disease, psychiatric history, prior 
therapies, hepatitis and HIV history), and any other assessments that are done for the purpose of determining 
eligibility for inclusion in the study. For women of childbearing potential, serum pregnancy test will be required at 
screening for eligibility and again at the end of the study. Urine pregnancy test may be used for onsite visits (if 
deemed clinically necessary). 
 
Physical examination must include a total body examination (general appearance, skin, neck, including thyroid, 
eyes, ears, nose, throat, lungs, heart, abdomen, back, lymph nodes, extremities and a basic nervous system 
neurological exam). Physical examinations will occur at the screening and month 6 (visit 8).  A physician will 
evaluate patient at each visit for significant changes and AEs per parent report. If noted, the physician will 
initiate a PRN physical exam as detailed above.  
 

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Significant findings made after the start of study drug that meet the definition of an Adverse Event must be 
recorded. 
 
 
7.23 GCP Statement 
 
This clinical trial will be conducted in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki, and that are consistent with Good Clinical Practice and all applicable regulatory 
requirements. 
 
8. Benefits 
 
Treatment on this study may or may not improve the cognitive and behavioral symptoms of individuals with 
PTEN gene mutation.  Information learned from this study may help patients with PTEN that are impacted by 
behavioral and cognitive dysfunctions by better describing the neurocognitive deficits and the safety and efficacy 
of available treatments.  
 
9. Risks 
 
Study Drug: This research study may involve unpredictable risks. Problems or side effects that are not now 
known could also occur. The participant will be given any new information that may affect their willingness to 
start or continue themselves or their child in the study. 
 
One of the most common possible side effects of the study drug, everolimus, are mouth lining changes 
(stomatitis) manifested as redness, irritation, swelling in the mouth or mouth ulcers (Section 6.3). 
 
Sample collection: there is a minor risk of pain, bleeding, bruising, or infection.  Any additional biological 
specimens to be used in future genetic and molecular studies will be obtained from skin or left-over tissue 
obtained from a clinically-indicated procedure.  Such procedures could include the minor risk of pain, bleeding, 
bruising, or infection 
 
Loss of Confidentiality: minor risk for a loss of confidentiality. Strict patient confidentiality will be observed 
throughout all aspects of the study. While medical records will be reviewed by members of the research team, 
no individually identifiable patient data will be distributed to non-research or care-giving team members. All data 
collected as a part of this study, with the exception of photos/videos, will be de-identified prior to being uploaded 
into our consortium database, or sent to another site. 
 
10. Study Records Retention 
 
Data will be stored for at least two years following study completion. 
 
11. Sample Destruction 
 
All specimens and the derivatives from them will be stored indefinitely unless the participant withdraws from the 
study and requests the destruction of samples in writing.  
 
12. Protocol Deviations 
 
Any deviations from the protocol will be logged at each individual site and reported to the central IRB (or local 
IRB for non-reliant sites) at time of continuing review. This information will also be captured on the protocol 
deviation CRF in the database.  Any significant deviations will be reported within 24 hours to the central IRB.   
 
13. Adverse Event Criteria and Reporting Procedures 
 
Information about all adverse events, whether volunteered by the subject, discovered by investigator 
questioning, or detected through physical examination, laboratory test or other means, will be collected and 
recorded and followed as appropriate. 
 

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13.1 Definitions and Reporting 
 
An adverse event is defined as the appearance of (or worsening of any pre-existing) undesirable sign(s), 
symptom(s), or medical condition(s) that occur after patient’s signed informed consent has been obtained.  
Abnormal laboratory values or test results occurring after informed consent constitute adverse events only if 
they induce clinical signs or symptoms, are considered clinically significant, require therapy (e.g., hematologic 
abnormality that requires transfusion or hematological stem cell support), or require changes in study 
medication(s). 
 
Conditions, or symptoms, that are present at the time of informed consent should be recorded on the Medical 
History CRF and be entered into the database. Adverse events that occur after informed consent should be 
recorded on the running AE log, as well as on the Adverse Events CRF and be entered into the database 
Adverse event monitoring should be continued for at least 28 days (or 5 half-lives, whichever is longer) following 
the last dose of study treatment. Adverse events (including lab abnormalities that constitute AEs) should be 
described using a diagnosis whenever possible, rather than individual underlying signs and symptoms. When a 
clear diagnosis cannot be identified, each sign or symptom should be reported as a separate Adverse Event. 
 
Adverse events will be assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) 
version 4. If CTCAE grading does not exist for an adverse event, the severity of mild, moderate, severe, and life-
threatening, corresponding to Grades 1 - 4, will be used. 
 
The occurrence of adverse events should be sought by non-directive questioning of the patient at each visit 
during the study. Adverse events also may be detected when they are volunteered by the patient during or 
between visits or through physical examination, laboratory test, or other assessments. As far as possible, each 
adverse event should be evaluated to determine: 
 
1. The severity grade (CTCAE Grade 1-4) 
2. Its duration (Start and end dates or if continuing at the Safety Follow-up Visit) 
3. Its relationship to the study treatment (Reasonable possibility that AE is related: No, Yes)  
4. Action taken with respect to study or investigational treatment (none, dose adjusted, temporarily interrupted, 
permanently discontinued, hospitalized, unknown, not applicable) 
5. Whether medication or therapy was given (no concomitant medication/non-drug therapy, concomitant 
medication/non-drug therapy) 
6. Outcome (not recovered/not resolved, recovered/resolved, recovering/resolving, recovered/resolved with 
sequela, fatal, unknown) 
7. Whether it is serious, where a serious adverse event (SAE) is defined as in 13.3.1. 
 
All adverse events should be treated appropriately. Such treatment may include changes in study drug 
treatment including possible interruption or discontinuation, starting or stopping concomitant treatments, 
changes in the frequency or nature of assessments, hospitalization, or any other medically required intervention. 
Once an adverse event is detected, it should be followed until its resolution, and assessment should be made at 
each visit (or more frequently, if necessary) of any changes in severity, the suspected relationship to the study 
drug, the interventions required to treat it, and the outcome. 
 
Information about common side effects already known about the investigational drug can be found in the 
Investigators’ Brochure (Appendix 1). This information should be included in the patient informed consent and 
should be discussed with the patient during the study as needed. Adverse event monitoring should be continued 
for at least 30 days following the last dose of study treatment. 
 
13.2 Laboratory Test Abnormalities 
 
Laboratory abnormalities that constitute an Adverse event in their own right (are considered clinically significant, 
induce clinical signs or symptoms, require concomitant therapy or require changes in study treatment), should 
be recorded on the Adverse Events CRF. Whenever possible, a diagnosis, rather than a symptom should be 
provided (e.g. anemia instead of low hemoglobin). Laboratory abnormalities that meet the criteria for Adverse 
Events should be followed until they have returned to normal or an adequate explanation of the abnormality is 
found. When an abnormal laboratory or test result corresponds to a sign/symptom of an already reported 
adverse event, it is not necessary to separately record the lab/test result as an additional event. 

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Laboratory abnormalities, that do not meet the definition of an adverse event, should not be reported as adverse 
events. A Grade 3 or 4 event (severe) as per CTCAE does not automatically indicate a SAE unless it meets the 
definition of serious as defined below and/or as per investigator’s discretion. A dose hold or medication for the 
lab abnormality may be required by the protocol and is still, by definition, an adverse event. 
 
In the event of a laboratory abnormality that the investigator believes to be inaccurate, repeat testing may be 
done at a facility geographically close to the participant and then shared with the study team. 
 
13.3 Serious Adverse Event Criteria and Reporting Procedures 
 
13.3.1 Definitions 
 
A serious adverse event (SAE) is defined as one which: 
 
Constitutes a congenital anomaly/birth defect 
 
Results in persistent or significant disability/incapacity 
 
Requires inpatient hospitalization or prolongation of existing hospitalization, unless hospitalization is for: 
o 
Routine treatment or monitoring of the studied indication, not associated with any deterioration 
in condition 
o 
Elective or pre-planned treatment for a pre-existing condition that is unrelated to the indication 
under study and has not worsened since signing the informed consent 
o 
Treatment on an emergency outpatient basis for an event not fulfilling any of the definitions of 
an SAE given above and not resulting in hospital admission 
o 
Social reasons and respite care in the absence of any deterioration in the patient’s general 
condition 
 
Is fatal or life-threatening 
 
Is medically significant i.e. defined as an event that jeopardizes the patient or may require medical or 
surgical intervention to prevent one of the outcomes listed above 
 
Adverse events that are grade 1-2 and expected should be reported through the RDCRN database within 20 
working days and logged appropriately. Determination of causality and relatedness should be made by the site 
PI.  
 
Adverse events that are grade 1-2 and unexpected should be reported through the RDCRN database within 24 
hours, to local IRBs, and to the sponsor only if the event suggests that there are greater risks to study subjects 
than previously suspected.  
 
Adverse events that are grade 3-4 should be considered serious if they fit into one of the categories listed above 
(as defined by the FDA).  If the event is classified as an SAE then the appropriate reporting guidelines must be 
followed. All SAEs will be further classified as serious adverse events or serious adverse drug reactions based 
on the events relation to the study drug. For SAEs, the final determination of causality is made by the Medical 
Review Officer. If the event is unrelated to the drug (probably not related or definitely not related) then it is 
defined as a Serious Adverse Event. If the event is related to the drug (definitely related, probably related, or 
possibly related) then it is defined as a serious adverse drug reaction. 
 
The principal investigator has the obligation to report all serious adverse events to the FDA, IRB, RDCRN, and 
Novartis Pharmaceuticals Drug Safety and Epidemiology Department (DS&E). 
 
All SAEs deemed reportable by the medical review officer, will be submitted to the FDA by the investigator 
utilizing the Form FDA 3500A (MedWatch Form). 
  
13.3.2 Adverse Event Reporting  
 
All adverse events will be recorded in the RDCRN database for reporting to respective IRBs, FDA, industry 
sponsors, and for data analysis. The DSC will review all AEs at regularly scheduled meetings. 
 

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 
Within 24 hours (of learning of the event), investigators must report any reportable Serious Adverse 
Event (SAE) that: 
o Is considered life-threatening/disabling or results in death of subject 
-OR- 
o Is Unexpected/Unanticipated  
 
Investigators must report all other reportable SAEs to the RDCRN database within 5 working days (of 
learning of the event) 
 
All other (suspected) reportable AEs must be reported to the RDCRN within 20 working days of the 
notification of the event or of the site becoming aware of the event. 
 
 
To ensure patient safety, every SAE, regardless of suspected causality, occurring after the patient has provided 
informed consent and until at least 28 days after the patient has stopped study treatment/participation, after 
protocol-specified procedures begin (e.g., placebo run-in, washout period, double-blind treatment, etc.) and 28 
days after the patient has stopped study treatment, after the start of any period in which the study protocol 
interferes with the standard medical treatment given to a patient (e.g., treatment withdrawal during washout 
period, change in treatment to a fixed dose of concomitant medication) and until 28 days after the patient has 
stopped study treatment. 
 
All SAEs must be reported to Novartis within 24 hours of learning of its occurrence.  Information about all SAEs 
is collected and recorded on the Serious Adverse Event Report Form; all applicable sections of the form must 
be completed in order to provide a clinically thorough report. The investigator must assess and record the 
relationship of each SAE to each specific study treatment (if there is more than one study treatment), complete 
the SAE Report Form in English, and send the completed, signed form by fax to (fax: 877-778-9739) within 
24 hours to the oncology Novartis DS&E department. Should the designated SAE Fax number be non-
functional please send SAEs to the designated SAE mailbox: clinicalsafetyop.phuseh@novartis.com. A Novartis 
SAE Coversheet must be attached to all SAE Submissions. This includes serious, related, not related, labeled 
(expected) and, unlabeled (unexpected) adverse experiences. All deaths during treatment or within 30 days 
following completion of active protocol therapy must be reported within24 hours. 
 
Any SAEs experienced after this 28 days period should only be reported to Novartis if the investigator suspects 
a causal relationship to the study drug. Recurrent episodes, complications, or progression of the initial SAE 
must be reported as follow-up to the original episode within 24 hours of the investigator receiving the follow-up 
information. A SAE occurring at a different time interval or otherwise considered completely unrelated to a 
previously reported one should be reported separately as a new event.  The end date of the first event must be 
provided. 
 
The original copy of the SAE Report and the fax confirmation sheet must be kept within the Trial Master File at 
the study site. 
 
Follow-up information is sent to the same fax number as the original SAE Report Form was sent, using a new 
fax cover sheet, stating that this is a follow-up to the previously reported SAE, and giving the date of the original 
report. Each re-occurrence, complication, or progression of the original event should be reported as a follow-up 
to that event regardless of when it occurs. The follow-up information should describe whether the event has 
resolved or continues, if and how it was treated, whether the blind was broken or not (if applicable), and whether 
the patient continued or withdrew from study participation. 
 
If the SAE is not previously documented in the Everolimus Investigator Brochure or Package Insert (new 
occurrence) and is thought to be related to the Novartis study drug, a DS&E associate may urgently require 
further information from the investigator for Health Authority reporting. Novartis may need to issue an 
Investigator Notification (IN), to inform all investigators involved in any study with the same drug that this SAE 
has been reported. Suspected Unexpected Serious Adverse Reactions (SUSARs) will be collected and reported 
to the competent authorities and relevant ethics committees in accordance with Directive 2001/20/EC or as per 
national regulatory requirements in participating countries. 
 
 
 
 

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13.4 Adverse Event Data Management  
 
Upon entry of a serious adverse event, the DMCC created Adverse Event Data Management Form will 
immediately notify the Study Chair, site PIs, the Medical Review Officer, and any additional agencies (if 
applicable - industry sponsor, CTEP, etc.) of any reported adverse events via email.  
 
Serious adverse events: The site physician will determine initial causality (definitely not related, probably not 
related, possibly related, probably related, definitely related) of the adverse event when submitting the report in 
the DMCC database. The final determination of causality will be made by the study appointed medical monitor, 
David Franz, M.D.. The medical monitor [and, if applicable, sponsor] may request further information if 
necessary and possibly request changes to the protocol or consent form as a consequence of the adverse 
event. A back-up notification system is in place so that any delays in review by the medical monitor beyond a 
specified period of time are forwarded to a secondary reviewer. The Adverse Event Data Management Form 
maintains audit trails and stores data (and data updated) and communication related to any adverse event in the 
study.  
 
Non-serious expected adverse events: Except those listed above as immediately reportable, non-serious 
expected adverse events that are reported to or observed by the investigator or a member of his/her research 
team will be submitted to the DMCC in a timely fashion (within 20 working days). The events will be presented in 
tabular form and given to the medical monitor and Safety Monitoring Committee on a bi-annual basis. Local site 
investigators are also required to fulfill all reporting requirements of their local institutions. 
 
The DMCC will post aggregate reports of all reported adverse events for site investigators and IRBs. 
 
13.5 Unanticipated Problem Reporting 
 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
subjects or others to include, in general, any incident, experience, or outcome that meets all of the following 
criteria: 
• unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and informed 
consent document; and (b) the characteristics of the subject population being studied; 
• related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• suggests that the research places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized. 
 
Per the definition, only a subset of adverse events would be characterized as unanticipated problems. There are 
other types of incidents, experiences, and outcomes that are not considered adverse events, but are 
characterized as unanticipated problems (e.g., breach of confidentiality or other incidents involving social or 
economic harm). 
 
Incidents or events that meet the OHRP criteria for unanticipated problems are to be reported to the IRB, per 
local institutional reporting requirements. Local institutional reporting requirements to IRBs, any GCRC oversight 
committee and the FDA, if appropriate, remain the responsibility of the treating physician and the Study Chair. 
 
13.6 Pregnancies 
 
Any pregnancy that occurs during study participation should be reported by the site. To ensure patient safety, 
each pregnancy must also be reported to Novartis within 24 hours of learning of its occurrence. The pregnancy 
should be followed up to determine outcome, including spontaneous or voluntary termination, details or birth, 
and the presence or absence of any birth defects, congenital abnormalities or maternal and newborn 
complications.  
 
Pregnancy should be recorded on a Clinical Study Pregnancy Form and reported by the investigator to the 
Novartis Drug Safety and Epidemiology (DS&E) department. Pregnancy follow-up should be recorded on the 

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same form and should include an assessment of the possible relationship to the Novartis study treatment of any 
pregnancy outcome. Any SAE experienced during pregnancy must be reported on the SAE Report Form.  
 
 
14. Study Oversight 
 
The study protocol will be reviewed and approved by the National Institutes of Health (NIH) before submission to 
individual center IRBs for approval.  Participant enrollment may only begin with IRB approved consent forms. 
 
Auditing procedures will be followed to ensure that the study is conducted, documented, and reported in 
accordance with the IRB approved protocol, Good Clinical Practice (GCP) Guidelines, and applicable regulatory 
requirements of collaborating sites. Verification of eligibility and appropriate documentation of informed 
consent/assent will be performed for a sample of subjects enrolled in the study. The investigator must maintain 
source documents for each patient in the study, consisting of case and visit notes (hospital or clinic medical 
records) containing demographic and medical information, laboratory data, and the results of any other tests or 
assessments. All information on CRFs must be traceable to these source documents in the patient's file. The 
investigator must also keep the original informed consent form signed by patient (a copy is given to the patient). 
 
15. Data and Safety Monitoring Committee (DSMC) 
 
The Study Chair has primary oversight responsibility of this clinical trial. The appointed Data and Safety 
Monitoring Committee (DSMC) has oversight responsibility of the Data and Safety Monitoring Plan (DSMP) for 
this clinical trial. Each site’s Principal Investigator and their research team (co-Investigators, research nurses, 
clinical trial coordinators, and data managers) are responsible for identifying adverse events. Aggregate report - 
detailed by severity, attribution (expected or unexpected), and relationship to the study drug/study procedures – 
will be available from the DMCC for site review.  Adverse events will be reviewed by the research team at time 
of occurrence and reviewed at monthly team meetings. 
 
The DSMC is an internally appointed group of individuals who will monitor and evaluate the safety of the 
proposed clinical trial. The DSMC’s primary role is to ensure the safety of patients in the study. The DSMC will 
follow the NIH's published guidelines regarding its function and may provide input such as the recommendation 
to stop the trial for safety reasons. Due to the signal-seeking nature and the small sample size in this study, 
there is no formal efficacy interim analysis planned. However, the DSMC will review adverse events and serious 
adverse events and provide a report to the group bi-annually. The DSMC will include individuals familiar with 
everolimus administration and clinical trial design and will be comprised of Phillip Pearl, M.D. (pediatric 
neurologist from Boston Children’s Hospital), Brenda Porter, M.D. (pediatric neurologist at Stanford University, 
Chairperson), and Eric Youngstrom, Ph.D. (biostatistician from the University of North Carolina).  
 
David Franz, M.D. (Cincinnati Children’s Hospital Medical Center) will be the independent medical review officer 
for this trial.  He is an expert in the use of mTOR inhibitors in children and adults with TSC. Dr. Franz will review 
any adverse events/serious adverse events throughout and will also determine if unblinding and reporting to the 
FDA and DSMC is required. If Dr. Franz is unable to fulfill his duty at any point during the study, Sarah Spence, 
M.D. (Boston Children’s Hospital) will serve as the back-up medical review officer until Dr. Franz returns to his 
duties. 
 
16. Data Management and Coordinating Center and Database 
 
All study data will be collected via systems created in collaboration with the Rare Diseases Clinical Research 
Network Data Management Coordinating Center (RDCRN DMCC) and will comply with all applicable guidelines 
regarding patient confidentiality and data integrity.  
 
16.1 Registration 
 
Registration of participants on this protocol will employ an interactive data system in which the clinical site will 
attest to the participant’s eligibility as per protocol criteria and obtain appropriate informed consent. IRB approval 
for the protocol must be on file at the DMCC before accrual can occur from the clinical site. 
 

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The DMCC will use a system of coded identifiers to protect participant confidentiality and safety.  Each 
participant enrolled will be assigned a local identifier by the enrollment site.  This number can be a combination 
of the site identifier (location code) and a serial accession number.  Only the registering site will have access to 
the linkage between this number and the personal identifier of the subject.  When the participant is registered to 
participate in the study, using the DMCC provided web-based registration system; site personnel will insert the 
unique ID generated from the National Database for Autism Research (NDAR) Global Unique Identifier (GUID) 
software program, explained in the following section 16.2, as the participant ID number.  Thus each participant 
will have two codes: the local one that can be used by the registering site to obtain personal identifiers and a 
second code designated by the NDAR GUID.  For all data transfers to the DMCC both numbers will be required 
to uniquely identify the subject.  In this fashion, it is possible to protect against data keying errors, digit 
transposition or other mistakes when identifying a participant for data entry since the numbers should match to 
properly identify the participant.  The only personal identifier accessible to the DMCC is participants’ date of 
birth, as well as the date of onset for all recorded medical events. The database will not collect any other 
components of a participant’s protected health information.   
 
If the subject is participating in more than one DSC study, such as this study and the DSC “Natural History 
Study of Individuals with Autism and Germline Heterozygous PTEN Mutations” study, data from those studies 
can be linked. 
 
16.2 Data Elements 
 
Every individual who provides consent will be assigned a Global Unique Identifier (GUID). The GUID is a unique 
universal subject ID that allows researchers to share data specific to a study participant without exposing 
personally identifiable information. A member of the research team will enter private health information (PHI) 
collected from each study participant into the National Database for Autism Research (NDAR) GUID software 
program. The software performs a one-way encryption of these data, de-identifying and randomizing the data to 
produce a digital “fingerprint”. The GUID program at NIH converts this digital fingerprint into a final GUID that is 
completely devoid of PHI. The final GUID is returned to the research team as a unique identifier for the 
participant. 
 
16.3 Data Entry 
 
Data collection for this study will be accomplished with online electronic case report forms.  Using encrypted 
communication links, online forms will be developed that contain the requisite data fields.  The DMCC, in 
collaboration with DSC, is also responsible for data sharing with the NIH National Database for Autism 
Research (NDAR) and the database of Genotypes and Phenotypes (dbGaP).  
 
All dosing data will be entered into the DMCC database within 24 business hours of the study visit to ensure the 
most up-to-date information is available for dose modification and participant safety decisions. 
 
16.4 Laboratory Data Flow 
 
Samples collected as a part of this study will be tracked utilizing the corresponding data collection sheets. These 
forms will be kept both locally and in the database to ensure that samples are sent to the appropriate 
laboratories. Samples results will be reported electronically to study sites directly from LabConnect.  
Additionally, the DMCC will data transfer from LabConnect electronically on a regular basis.  All transactions are 
logged and validated for both methods. 
 
16.5 Data Quality and Monitoring Measures 
 
As much as possible data quality is assessed at the data entry point using intelligent on-line data entry via visual 
basic designed screen forms. Data element constraints, whether independent range and/or format limitations or 
‘relative’ referential integrity limitations, can be enforced by all methods employed for data input. QA reports 
assess data quality post-data entry. As we note, data quality begins with the design of the data collection forms 
and procedures and incorporates reasonable checks to minimize transcription and omission errors.  Of the more 
important quality assurance measures are the internal validity checks for reasonableness and consistency.  
• Data Monitoring: The RDCRN DMCC will utilize the Data Quality Module within REDCap to identify 
missing or unclear data. Study sites will be able to generate queries and resolve for their site.  

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• Data Delinquency Tracking: REDCap will allow the consortium sites to monitor data delinquency on an 
as needed basis. 
 
16.6 Quality Control Method 
 
Standardized data collection and management procedures will be followed to ensure data quality control. Data 
will be scored, reduced, and managed on an ongoing basis. The PIs will assume responsibility for uniformity 
across sites and will coordinate on a weekly basis (or more frequently if needed) with the data collection and 
management team. After data have been collected, it will be entered into the RDCRN DMCC database. As 
much as possible, data quality is assessed at the data entry point using intelligent on-line data entry via visual 
basic designed screen forms. Data element constraints, whether independent range and/or format limitations or 
‘relative’ referential integrity limitations, can be enforced by all methods employed for data input. QA reports 
assess data quality post-data entry. As we note, data quality begins with the design of the data collection forms 
and procedures and incorporates reasonable checks to minimize transcription and omission errors.  Of the more 
important quality assurance measures are the internal validity checks for reasonableness and consistency. 
 
All variables will be checked for out-of-range values and the marking of missing values.  
 
17. Data Analysis Plan, Statistical Power and Sample Considerations  
 
This is an exploratory study to evaluate the short-term safety and preliminary efficacy of everolimus in patients 
with a PTEN mutation. The analyses will be predominantly descriptive with the intent of generating plausible 
hypotheses to be tested in a Phase III confirmatory trial. Efficacy will be assessed using recognized measures of 
intellectual ability and neurobehavioral processes.  
 
In objective 1, we will examine the safety and tolerability of everolimus by comparing the 1) dropout rates due to 
side effects, 2) dropout rates for any reason, and the 3) frequency of specific side effects in those receiving 
everolimus versus placebo. Based on the published literature and our experience in the tuberous sclerosis trial, 
we predict that the dropout rates due to side effects will be very similar across everolimus and placebo groups 
(<10% difference). However, we predict that everolimus will cause higher rates of medication-related side 
effects (≥10% difference) when compared to placebo. We will use chi-square statistics (or Fisher’s exact test 
depending on cell counts) to compare dropout and rates of specific side effects. Statistical significance will be 
determined using a one-tailed test Type 1 error rate of α=.05, as only increases with everolimus treatment are 
expected. No false discovery rate correction will be applied because the concern is maximizing sensitivity to any 
increase in side effect prevalence with everolimus treatment. For dropout rates, we will also compute Kaplan-
Meier survival analyses with discontinuation due to side effects or any reason as separate status endpoints. 
Number of days from baseline to medication discontinuation will be the time variable. Survival analyses are 
expected to be under-powered due to the generally low dropout rates expected, but these analyses can be 
useful for describing temporal trends. To ensure any observed group differences are not influenced by other 
variables or randomization imbalance, cox and logistic regression analysis will also be conducted with and 
without conditioning on relevant baseline covariates (e.g., demographics, language and cognitive ability, 
symptom severity). 
 
In objective 2, we will examine whether the everolimus group will show more improvement, compared to the 
placebo group, on the main neurocognitive efficacy composite outcome. Randomization Group (everolimus 
versus placebo) differences on the neurocognitive composite will be analyzed using mixed effects (growth 
curve) modeling, fully utilizing repeated measurements collected at three points (Time: baseline, 3-month follow-
up, and 6-month follow-up). A significant interaction between Time and Randomization Group, with the 
everolimus group showing a more favorable outcome trajectory, will support the primary efficacy hypothesis. 
Maximum likelihood estimation will be implemented in the Mplus program version 7.2 or above.  
 
Mixed effects (growth curve) model analyses will be conducted following the intention-to-treat principle. Data 
points that are missing due to subject attrition will be handled assuming that data are missing at random (MAR) 
conditional on observed information, which is less restrictive than missing completely at random (MCAR) 
assumed in fixed effects analyses such as ANCOVA and regression analysis. In this procedure, all available 
cases including the ones with missing information will be included in the analyses. By including every subject 
who completed at least one assessment, we are not only more likely to conserve power, but also less likely to 
produce biased effect estimates. In mixed effects analyses, the slope of the outcome will be modeled as the key 

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dependent variable predicted by randomization group. We will conduct the analysis with and without 
conditioning on relevant baseline covariates. The results of these longitudinal analyses can be easily converted 
to a cross-sectional group effect at each assessment time point to describe the magnitude and significance of 
group differences. Of particular interest is the group difference at end of treatment (6-month assessment). The 
mixed effects analyses will be repeated using a model where the baseline scores on each outcome are treated 
as a baseline covariate to control for (instead of as a part of) the repeated measures. This model can be useful if 
there is a considerable heterogeneity between before and after treatment processes. We will also analyze the 
data using conventional ANCOVA treating the end point neurocognitive composite as the dependent variable 
and controlling for the baseline neurocognitive composite. Further analyses will be conducted using the same 
analysis models but with transformed data (e.g., log transformation) to check the sensitivity of the results to 
deviation from outcome normality. Statistical significance of all main effects and interaction terms as well as 
cross-sectional group differences will be determined using a one-tailed Type 1 error rate of α=.05. The same 
analysis strategy will be employed for secondary efficacy outcomes and a false discovery rate correction will be 
implemented to control inflation of Type 1 error. 
 
If the exploratory biosample objectives are pursued, we will explore whether baseline peripheral blood levels of 
PTEN-associated pathway molecules (PI3K/AKT, mTOR, MAPK protein levels) change with treatment and if 
changes in peripheral pathway molecules correlate with clinical improvement. To determine whether PTEN-
associated pathway molecules change with treatment, we will use a similar mixed effect (growth curve) 
modeling approach to that described above for Secondary Objective with screening, 3-month, and 6-month 
pathway measurements as outcome measures. The Time by Randomization Group interaction will test whether 
the everolimus treatment group shows improvement/normalization of PTEN-pathway molecule levels relative to 
placebo. This component will be dependent on securing additional funds.  
 
In analyzing the resting state EEG and AEP data we will focus on the following: 
1. Baseline EEG gamma power 
2. AEP N1 habituation 
3. AEP variability (phase-locking factor, also called intertrial phase coherence) 
 
EEG and AEP analysis will occur via the Boston EEG Automated Processing Pipeline (BEAPP), created at 
Boston Children’s Hospital by April Levin MD.  BEAPP is MATLAB-based software designed to facilitate batch 
processing of EEG files for artifact removal and signal processing in an automated manner.  Users have the 
option to alter multiple settings, to customize processing to their needs.  The pipeline is freely available and 
covered under a General Public License, which allows other groups to replicate or alter analyses for their own 
data if they wish. 
 
To prepare EEG and AEP data for analysis, the pipeline imports EEG from various acquisition formats and 
electrode layouts into a standardized format. Data will first be processed via PREP, which removes 60 Hz line 
noise, identifies and interpolates bad channels, and references to average.58  Data will then be high pass filtered 
at 1 Hz, and will undergo artifact removal via MARA, an automated independent component-based supervised 
machine learning algorithm that handles artifacts including ocular artifact, muscle artifact, and loose electrodes.  
 
For analysis of resting EEG data, segments of high-amplitude artifact (>150 uV) will be removed, and the 
remaining data will be segmented into 2-second epochs.  Data will undergo a Laplacian transform, as this has 
been shown to reduce sensitivity of the EEG signal to contamination by myogenic activity, which is otherwise 
particularly prominent in the gamma band.59,60  Spectral analysis will be performed on each epoch with 
multitaper methods using 3 tapers, in order to determine the average power spectrum across all epochs.  This 
will allow for determination of power in the gamma frequency band, particularly the 62-90Hz range. 
 
For analysis of AEP data, data will be epoched into 2000 ms trials (-500 to 1500 ms), and baseline corrected 
using the 500 ms period prior to the first auditory stimulus in each trial.  Any trial with amplitude exceeding 150 
uV will be removed.  N1 will be defined as the most negative-going waveform deflection between 50 and 150ms 
post-stimulus, over the central region.  (If N1 cannot be adequately identified in this population using these 
criteria, independent components analysis will be used to identify the N1 component instead).  Habituation of 
the N1 amplitude will be quantified as percent change from the first to second auditory click in each trial.  
Intertrial phase coherence (ITPC) will be calculated using the function newtimef, and reported as the maximum 
absolute value ITPC in the 50-150ms timeframe, in the alpha frequency band (8-12Hz). 
 

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Two approaches will be used to explore whether changes in PTEN-pathway molecules are correlated with 
changes in clinical improvement. First, we will use the mixed effects modeling framework to examine the 
correlation between the longitudinal changes in the neurocognitive composite score and changes in PTEN-
associated pathway molecules. In this framework, the relationship between slopes representing individual 
clinical changes and individual PTEN-pathway molecule changes are estimated. We will also examine whether 
this correlation differs by treatment group. Second, we will estimate a structural model with cross-lagged 
relationships estimated between screening/3-month PTEN-pathway levels and 3-month/6-month efficacy 
measurements. Rather than evaluating the relationship between changes over time, the cross-lagged structural 
model examines whether pathway molecule levels at earlier time points predict efficacy measurements at later 
time points. Each of these methods will use all available data following the intent-to-treat principle. As a result, 
they are less likely to produced biased estimates than a bivariate correlation approach using changes from 
baseline because the latter method can only be computed for individuals with complete data. 
 
17.1 Power Estimation 
 
This is the first study to evaluate the efficacy of everolimus in patients with a PTEN mutation and ASD/DD. As 
such, analyses will be predominantly descriptive with the intent of generating plausible hypotheses to be tested 
in a Phase III confirmatory trial. The planned recruitment sample size of 40 was based on three considerations: 
1) the expected recruitment potential of the three sites, 2) a desire to ensure sensitivity to any enrichment in side 
effects or problems with tolerability in everolimus treated patients, and 3) the need for adequate statistical power 
in examining primary and secondary efficacy outcomes. Assuming an overall dropout rate of approximately 
10%, 44 patients will need to be enrolled to get 40 patients with complete trial data. However, following intent to 
treat procedures, all patients who are randomized, dosed, and for whom baseline data is collected will be 
included in statistical analyses. Statistical power considerations for each aim/hypothesis are described below. 
Except for safety analyses, where only everolimus-treated patients would be expected to show enrichment of 
dropout or side effects, statistical power estimates are based on one-tailed significance tests with Type 1 error 
rate of α=.05. 
 
Primary Objective- Safety. The planned sample size of 40 is underpowered to detect small differences in 
dropout rates or side effects between everolimus versus placebo-treated groups. Statistical power will only be 
adequate if at least a 35% difference in the proportion of dropout/side effects is observed (1-β≥.81), assuming a 
one-tailed Type 1 error rate of α=.05. This is equivalent to 1/20 patients experiencing an adverse event/side 
effect in the placebo arm and 8/20 in the treated arm. Similarly, statistical power of survival analyses will only be 
fair (1-β≥.57) if a hazard ratio of 7.5 is observed, equivalent to a 5% dropout rate in the placebo group versus a 
30% dropout rate in the everolimus group. However, because the goal is to maintain sensitivity to any potential 
enrichment in dropout or side effects, any indicator showing a difference of 10% or greater will be considered a 
meaningful difference and will be reported. 
 
Secondary Objective – Efficacy. For power estimation purposes, the variability in PTEN patients treated with an 
mTOR inhibitor is expected to be similar to the untreated sample. Preliminary data indicates that the measures 
which comprise the dominant portion of the neurocognitive composite index (processing speed working 
memory, and fine motor) are highly sensitive to the brain dysfunction seen in PTEN patients with ASD. The 
effect sizes are approximately 1 SD or larger. In a comparison between mTOR treated vs. untreated PTEN 
patients, an effect size of at least 0.65 SD would be of interest and represent clinically- meaningful 
improvement. For example, an effect size of at least 0.65 SD on cognitive measures would translate to a 10-
point standard score improvement in the everolimus-treated group relative to placebo. An improvement of 10 or 
more points is outside the standard error of measurement for most cognitive measures and is typically 
considered a reliable change when disordered populations are re-tested.   
 
A sample size of 40 (20 per arm) patients will permit detection of endpoint (6-month follow-up) differences of at 
least 0.80 SD at the 5% significance level (one-sided alpha = 0.05) with 80% power (1-β). Statistical power will 
be weaker (1-β=.65) if smaller cross-sectional differences are observed (0.65 SD). However, statistical power is 
very strong, even for smaller differences (Cohen’s d≥.50 equivalent to ≥.50 SD difference) when the full 
repeated measures nature of the design is considered – baseline, 3-month follow-up, 6-month follow-up. 
Assuming even modest correlations between repeated measurements of outcome variables (r≥.30), power to 
detect clinically meaningful differences of .50 SD is very good (1-β=.83; one-sided α=.05). If the primary 
outcome measure (neurocognitive composite) meets nominal statistical significance in the expected direction 
(one-tailed p<.05) or a secondary outcome measure meets a more stringent false discovery rate-corrected 

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significance level in the expected direction, these outcomes will be considered candidates for Phase III 
confirmatory trial evaluation. To determine if a secondary outcome meets the more stringent false discovery 
rate-corrected significance level, a Benjamini-Hochberg false discovery rate correction will be applied with 
adjustment for all secondary outcome measures. This adjustment involves sorting the list of obtained p-values in 
descending order. The smallest p-value is then compared to the most stringent alpha, the next smallest is 
compared to the next most stringent alpha, and so on. Only those secondary outcomes meeting FDR-corrected 
alpha level will be considered candidates for Phase III evaluation.  
 
An interim analysis will be conducted after half the sample has been collected (10 patients in each arm) and will 
mainly focus on safety. Efficacy interim analysis may be under-powered, but will focus on whether the effects 
are in the expected direction and whether effect sizes (both the endpoint effect size and the interaction fully 
utilizing repeated measures) are approaching expected levels (at least Cohen’s d≥.30). The goal of the interim 
analysis for efficacy will be to get a signal that will allow us to design more targeted trials in the future. As such, 
we will check for a signal in any domain and, if needed, include additional endpoints before the end of the study 
to expand the endpoint and optimize outcome. 
 
Additional considerations. This is the first clinical trial in patients with PTEN mutations and ASD/DD. For this 
reason, and to facilitate recruitment, all types of PTEN mutations will be permitted within the randomized 
patients. However, post-hoc analyses will be completed to examine the existence of any differential effect of 
treatment based on the type of PTEN mutation (missense vs. other mutation types) or mutation location (exon 
number, location in 3-D PTEN structure).  
 
18. Protocol Amendments or Changes in Study Conduct 
 
Any change or addition (excluding administrative) to this protocol requires a written protocol amendment that 
must be reviewed by the central IRB at Boston Children’s Hospital (Boston and Stanford only), Cleveland Clinic 
IRB the NIH, and Novartis as applicable.  Amendments significantly affecting the safety of subjects, the scope of 
the investigation or the scientific quality of the study require additional approval by the IRB at each study center.  
A copy of the written approval of the IRB must be provided to Novartis.  
 
Examples of amendments requiring such approval are: 
1. increases in drug dose or duration of exposure of subjects, 
2. significant changes in the study design (e.g. addition or deletion of a control group), 
3. increases in the number of invasive procedures, 
4. addition or deletions of a test procedure required for monitoring of safety. 
 
These requirements for approval should in no way prevent any immediate action from being taken by the 
investigator or by Novartis in the interests of preserving the safety of all patients included in the trial.  If an 
immediate change to the protocol is felt to be necessary by the investigator and is implemented for safety 
reasons Novartis must be notified and the IRB at the center must be informed immediately.   Amendments 
affecting only administrative aspects of the study do not require formal protocol amendments or IRB approval 
but the IRB must be kept informed of such administrative changes. Examples of administrative changes not 
requiring formal protocol amendments and IRB approval include: 
1. 
changes in the staff used to monitor trials  
2. 
minor changes in the packaging or labeling of study drug. 
 
19. Compensation 
 
As a part of this study we may be able to provide travel reimbursement (i.e. airfare, hotel, and mileage costs) 
and/ or parking and meal vouchers for participants. The reimbursement process at Boston Children’s Hospital 
and Stanford University will go through the National Organization for Rare Disorders (NORD). NORD will 
provide reimbursements in the form of a check to families after they have completed the study visit. The study 
team at Cleveland Clinic will reimburse their participants directly. Participants/families can receive up to $1,500 
per completed study visit. 
 
20. Privacy Provisions 
 

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To maintain the privacy of potential subjects, they will only be approached regarding study enrollment as part of 
their routine clinical care or via a mailed letter that they may choose to response to. Individuals will not be 
approached by someone they do not know or anticipate in an unknown or unwanted environment. Data and 
identifying information collected as part of this study will only be released to those listed in the consent/assent 
forms.  
 
21. Confidentiality Provisions 
 
Subjects will be provided with a unique ID code and only the study coordinator, Principal Investigator and study 
monitor will be able to view the link between participants’ names and the unique ID. The link to all codes will be 
maintained in a secure, HIPAA compliant database in the study coordinator’s office. The unique patient number 
will be used on all research documents, including case report forms, as well as on labels on samples collected. 
All information from this study will be kept in the same research binder in the study coordinator’s private and 
locked office. This will include all paperwork with identifying information, including tracking sheets, orders, 
consents, etc.  
 
Subject confidentiality will be maintained by the investigator, the investigator’s associates and co-workers, and 
by all administrators who are part of the project.  Confidentiality will be maintained according to ICH E6; 4.8.10, 
part O: “Records identifying the subject will be kept confidential and, to the extent permitted by the applicable 
laws and/or regulations, will not be made publicly available.  If the results of the trial are published, the subject’s 
identity will remain confidential.” 
 
The investigator, his or her staff and associates, and the appropriate regulatory agencies may use the 
information included in this protocol as necessary for the conduct of the trial and the safety of subjects.  The 
parent/guardian/subject may obtain the results of clinical and procedures obtained for this research study if (1) 
results are available and (2) disclosure does not have the potential to impact the accuracy of future 
assessments of the subject during the course of the study.   
 
Data file records that are to be shared between sites will be encrypted before sharing so that they will be 
undecipherable if intercepted in transit.  All subject data entered into the study database will be identified only by 
a unique identifier for research records. Publications will reflect only unique identifiers. Data on paper will be 
kept locked.  Any data on computer will be accessible only by password access.  Only members of the research 
team will have access to these files.  
 
In addition, data will be placed into one or more publicly-accessible scientific databases.  For example, the 
National Institutes of Health maintained databases called the database of Genotypes and Phenotypes (dbGaP) 
and the National Database for Autism Research (NDAR). 
 
Data from this study will be submitted to the National Database for Autism Research (NDAR).  NDAR is a 
computer system run by the National Institutes of Health that allows researchers studying autism to collect and 
share information with each other.  With an easier way to share, researchers hope to learn new and important 
things about autism more quickly than before.  
 
During and after the study, the researchers will send information obtained about the subject’s health, behavior 
and genetics information to NDAR and/or submit to other publicly-accessible scientific databases.  However, 
before the information is sent, the researchers will remove information such as name, address, and phone 
number, and replace that information with a code number.  Other researchers nationwide can then file an 
application with the National Institutes of Health to obtain access to the study data for research purposes.  
Experts at the National Institutes of Health who know how to protect health and science information will look at 
every request carefully to minimize risks to subject privacy. 
 
21.1 Certificate of Confidentiality  
 
This research is covered by a Certificate of Confidentiality from the National Institutes of Health. The 
researchers cannot be forced to disclose information that may identify a study participant, even by a court 
subpoena, in any federal, state, or local civil, criminal, administrative, legislative, or other proceedings.  The 
researchers will use the Certificate to resist any demands for information that would identify a participant, except 
as explained below. 

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The Certificate cannot be used to resist a demand for information from personnel of the United States 
Government that is used for auditing or evaluation of Federally funded projects or for information that must be 
disclosed in order to meet the requirements of the federal Food and Drug Administration (FDA). 
 
Even with the Certificate of Confidentiality, the investigators continue to have ethical obligations to report child 
abuse or neglect and to prevent an individual from carrying out any threats to do serious harm to themselves or 
others.  If keeping information private would immediately put the study participant or someone else in danger, 
the investigators would release information to protect the participant or another person. 
 
Department of Health and Human Services (DHHS) personnel may request identifying information for purposes 
of performing audits, carrying out investigations of DHHS grant recipients, or evaluating DHHS funded research 
projects. 
 
 
 

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1. 
Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second 
messenger, phosphatidylinositol 3,4,5-trisphosphate. Journal of Biological Chemistry. 1998;273(22):13375-
13378. 
2. 
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