Phase I Study of MLN0128 (TAK-228) (NSC# 768435) in Combination With Ziv-Aflibercept (NSC# 724770) in Patients With Advanced Cancers
Phase: PHASE1
Conditions: Advanced Malignant Solid Neoplasm, Fibrolamellar Carcinoma, Metastatic Malignant Solid Neoplasm, Ovarian Carcinoma, Pancreatic Neuroendocrine Tumor, Recurrent Malignant Solid Neoplasm, Refractory Malignant Solid Neoplasm, Unresectable Solid Neoplasm
Sponsor: National Cancer Institute (NCI)

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NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
 
SUMMARY OF CHANGES - PROTOCOL 
 
For Protocol Amendment # to: Phase I Study of MLN0128 (TAK-228) (NSC# 768435) in 
Combination with Ziv-Aflibercept (NSC# 724770) in Patients with Advanced Cancers 
 
NCI Protocol #: 9585 
Local Protocol #: 2013-0665 
 
NCI Version Date: October 8, 2019 
Protocol Date:  October 8, 2019 
 
I. Protocol Changes by Rapid Amendment (Amendment 16/ Version 10; 07Oct2019) 
# 
Section 
Page 
Comments 
1. 
Throughout 
and Title Page 
 
All 
PI RESPONSE: The protocol version and version date was 
updated throughout the protocol on the title page and headers. 
Old Text:  
NCI Version Date:  June 11, 2019 
Protocol Date: June 11, 2019 
Protocol Type / Version # / Version Date 
Original/ Local Version #00 / February 24, 2014 
Amendment 01/ Local Version #01 / March 28, 2014 
Amendment 02/ Local Version #02 / August 15, 2014 
Amendment 03/ Local Version #03 / October 21, 2014 
Amendment 04/ Local Version #03 / November 7, 2014 
Amendment 05/ Local Version #03 / February 2, 2015 
Amendment 06/ Local Version #03 / March 20, 2015 
Amendment 07/ Local Version #04 / October 6, 2015 
Amendment 08/ Local Version #05 / January 5, 2016 
Amendment 09/ Local Version #06 / November 2, 2016 
Amendment 10/ Local Version #07 / August 3, 2017 
Amendment 11/ Local Version #07/ September 21, 2017 
Amendment 12/ Local Version #07/ December 22, 2017 
Amendment 13/ Local Version #07/ January 29, 2018 
Amendment 14/ Local Version #08/ June 20, 2018 
Amendment 15/ Local Version #09/ June 11, 2019 
New Text: 
NCI Version Date:  October 8, 2019 
Protocol Date: October 8, 2019 
Protocol Type / Version # / Version Date 
Original/ Local Version #00 / February 24, 2014 

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Amendment 01/ Local Version #01 / March 28, 2014 
Amendment 02/ Local Version #02 / August 15, 2014 
Amendment 03/ Local Version #03 / October 21, 2014 
Amendment 04/ Local Version #03 / November 7, 2014 
Amendment 05/ Local Version #03 / February 2, 2015 
Amendment 06/ Local Version #03 / March 20, 2015 
Amendment 07/ Local Version #04 / October 6, 2015 
Amendment 08/ Local Version #05 / January 5, 2016 
Amendment 09/ Local Version #06 / November 2, 2016 
Amendment 10/ Local Version #07 / August 3, 2017 
Amendment 11/ Local Version #07/ September 21, 2017 
Amendment 12/ Local Version #07/ December 22, 2017 
Amendment 13/ Local Version #07/ January 29, 2018 
Amendment 14/ Local Version #08/ June 20, 2018 
Amendment 15/ Local Version #09/ June 11, 2019 
Amendment 16/ Local Version #10/ October 8, 2019 
 
2. Collaborators 
3 
PI RESPONSE: As part of a minor administrative change, 
the study personnel have been updated. 
Old Text:  
Statistician: 
Kenneth Hess, PhD 
1515 Holcombe Blvd., Unit 1411 
Houston, Texas 77030 
713-794-4168 
khess@mdanderson.org 
Study Coordinator:  
Dipti Jain 
1515 Holcombe Blvd., Unit 455 
Houston, Texas 77030 
713-792-1469 
713-606-3912 
djain@mdanderson.org 
Responsible Research Nurse: 
Valerie Marcott, RN 
1515 Holcombe Blvd., Unit 455 
Houston, Texas 77030 
713-794-1014 
713-404-1550 
VDMarcot@mdanderson.org 

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New Text: 
Statistician: 
Suyu Liu, PhD 
1515 Holcombe Blvd., Unit 1409 
Houston, Texas 77030 
713-563-4280 
syliu@mdanderson.org 
Study Coordinator:  
Fengying “Fiona” Ouyang 
1515 Holcombe Blvd., Unit 455 
Houston, Texas 77030 
713-792-7192 
713-404-5990 
Fouyang@mdanderson.org 
Responsible Research Nurse: 
Jing Gong 
1515 Holcombe Blvd., Unit 455 
Houston, Texas 77030 
713-563-8392 
713-606-3681 
jinggong@mdanderson.org 
3. 7.1 CAEPRs for 
CTEP IND 
Agent(s) 
 
55 
PI RESPONSE: Per the Request a Rapid Amendment (RRA) 
for this NCI-CTEP trial, we have updated the protocol 
MLN0128 (TAK-228) risks based on the Comprehensive 
Adverse Events and Potential Risks (CAEPR) list (Version 
2.3, July 28, 2019). This section now utilizes CTCAE 5.0 
language, which is consistent with all other sections of the 
protocol.   
• The SPEER grades have been updated. 
• The section below utilizes CTCAE 5.0 language unless 
otherwise noted. 
 
• Added New Risk:  
• Also Reported on MLN0128 Trials But With Insufficient 
Evidence for Attribution: Abdominal distension; Abdominal 
infection; Acidosis; Alkaline phosphatase increased; 
Alopecia; Ataxia; Blood and lymphatic system disorders - 
Other (hyperviscosity syndrome); Blood and lymphatic 
system disorders - Other (Raynaud's phenomenon); Blood 

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lactate dehydrogenase increased; Bone pain; Chest wall pain; 
Colitis; Febrile neutropenia; Flu like symptoms; Fracture; 
Gastritis; Gastrointestinal disorders - Other (intestinal 
perforation); Gastrointestinal disorders - Other (salivary 
hypersecretion); General disorders and administration site 
conditions - Other (groin pain); GGT increased; 
Hemorrhoids; Hiccups; Hyperkalemia; Hypernatremia; 
Hypertension; Hyperuricemia; Ileus; Infections and 
infestations - Other (cystitis); Infections and infestations - 
Other (lower respiratory tract infection); Infections and 
infestations - Other (mucosal infection); Infections and 
infestations - Other (parotid gland); Injury, poisoning and 
procedural complications - Other (accidental overdose); 
Injury, poisoning and procedural complications - Other 
(postoperative fever); Injury, poisoning and procedural 
complications - Other (subdural hemorrhage); Lethargy; 
Lipase increased; Malaise; Metabolism and nutrition 
disorders - Other (severe chronic malnutrition); Neoplasms 
benign, malignant and unspecified (incl cysts and polyps) - 
Other (non-hodgkin lymphoma); Nervous system disorders - 
Other (neuropathy peripheral); Papulopustular rash; 
Paresthesia; Pleuritic pain; Proteinuria; Radiculitis; Renal 
and urinary disorders - Other (strangury); Stroke; Vaginal 
hemorrhage; Ventricular arrhythmia 
• Increase in Risk Attribution:  
• Changed to Likely from Less Likely: Constipation 
• Changed to Less Likely from Also Reported on MLN0128 
Trials But With Insufficient Evidence for Attribution: Acute 
kidney injury; Dyspepsia 
 
• Provided Further Clarification: (e.g., changed from 
‘arrhythmia’ to ‘ventricular fibrillation’) 
• Respiratory, thoracic and mediastinal disorders - Other 
(oropharyngeal pain) is now reported as Oropharyngeal pain. 
• Eye disorders - Other (visual acuity reduced) is now reported 
as Vision decreased. 
• Musculoskeletal and connective tissue disorder - Other 
(muscle spasms) is now reported as Muscle cramp. 
• Personality change is now reported as Psychiatric disorders - 
Other (mental status changes). 
• Urinary tract pain is now reported as Dysuria. 
PLEASE NOTE:  The specific detailed changes listed here 
compare the new revised CAEPR Version 2.3, and associated 

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risk information for the ICD, to the most recent CAEPR Version 
2.2.   If your trial contains an older CAEPR version (i.e., does 
NOT currently contain CAEPR Version 2.2), you MUST include 
a description of any additional changes resulting from migration 
from the older CAEPR version. 
New Text: 
7.1.1.1  CAEPR for MLN0128 (TAK-228) (NSC# 768435)  
Version 2.3, July 28, 20191
 
 Adverse Events with Possible  
 Relationship to MLN0128 (TAK-228) 
 (CTCAE 5.0 Term) 
[n= 390] 
  
 
 Specific Protocol 
Exceptions to 
Expedited Reporting 
(SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious 
(<3%) 
  
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
   
 
Anemia 
 
  Anemia (Gr 2) 
CARDIAC DISORDERS 
   
 
 
Cardiac arrest 
   
 
 
Ventricular 
fibrillation 
   
GASTROINTESTINAL DISORDERS 
   
 
Abdominal pain 
 
  Abdominal pain (Gr 
2) 
Constipation 
 
 
  Constipation (Gr 2) 
Diarrhea 
 
 
  Diarrhea (Gr 2) 
 
Dry mouth 
 
  Dry mouth (Gr 2) 
 
Dyspepsia 
 
   
Mucositis oral 
 
 
  Mucositis oral (Gr 2) 
Nausea 
 
 
  Nausea (Gr 3) 
Vomiting 
 
 
  Vomiting (Gr 3) 
GENERAL DISORDERS AND ADMINISTRATION SITE 
CONDITIONS 
   
 
Edema limbs 
 
    
Fatigue 
 
 
  Fatigue (Gr 3) 
 
Fever 
 
  Fever (Gr 2) 
 
General disorders and 
administration site 
conditions - Other 
(mucosal 
inflammation) 
 
  General disorders and 
administration site 
conditions - Other 
(mucosal 
inflammation) (Gr 2) 
INFECTIONS AND INFESTATIONS 
   
 
Urinary tract 
infection 
 
  Urinary tract 
infection (Gr 2) 
INVESTIGATIONS 
   
 
Creatinine increased  
  Creatinine increased 
(Gr 2) 
 
 
Electrocardiogram 
QT corrected 
interval prolonged 
   
 
Platelet count 
decreased 
 
  Platelet count 
decreased (Gr 2) 
 
Weight loss 
 
  Weight loss (Gr 2) 
METABOLISM AND NUTRITION DISORDERS 
   
Anorexia 
 
 
  Anorexia (Gr 2) 
 
Dehydration 
 
  Dehydration (Gr 2) 

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Hyperglycemia 
 
 
  Hyperglycemia (Gr 3) 
 
Hypokalemia 
 
  Hypokalemia (Gr 2) 
 
Hypomagnesemia 
 
  Hypomagnesemia (Gr 
2) 
 
Hypophosphatemia 
 
  Hypophosphatemia 
(Gr 2) 
MUSCULOSKELETAL AND CONNECTIVE TISSUE 
DISORDERS 
   
 
Arthralgia 
 
   
 
Back pain 
 
  Back pain (Gr 2) 
 
Pain in extremity 
 
   
NERVOUS SYSTEM DISORDERS 
   
 
Dizziness 
 
  Dizziness (Gr 2) 
 
Dysgeusia 
 
  Dysgeusia (Gr 2) 
 
Headache 
 
  Headache (Gr 2) 
PSYCHIATRIC DISORDERS 
   
 
Insomnia 
 
   
RENAL AND URINARY DISORDERS 
   
 
Acute kidney injury  
   
RESPIRATORY, THORACIC AND MEDIASTINAL 
DISORDERS 
   
 
Cough 
 
  Cough (Gr 2) 
 
Dyspnea 
 
  Dyspnea (Gr 2) 
 
Oropharyngeal pain  
   
 
 
Pneumonitis 
   
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
   
Pruritus 
 
 
  Pruritus (Gr 2)  
Rash maculo-
papular 
 
 
  Rash maculo-papular 
(Gr 2) 
1This table will be updated as the toxicity profile of the agent 
is revised.  Updates will be distributed to all Principal 
Investigators at the time of revision.  The current version can 
be obtained by contacting PIO@CTEP.NCI.NIH.GOV. Your 
name, the name of the investigator, the protocol and the 
agent should be included in the e-mail. 
Adverse events reported on MLN0128 (TAK-228) trials, but 
for which there is insufficient evidence to suggest that there 
was a reasonable possibility that MLN0128 (TAK-228) 
caused the adverse event: 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Blood 
and lymphatic system disorders - Other (hyperviscosity 
syndrome); Blood and lymphatic system disorders - Other 
(Raynaud's phenomenon); Febrile neutropenia 
CARDIAC DISORDERS - Heart failure; Pericardial 
effusion; Sinus tachycardia; Ventricular arrhythmia 
EYE DISORDERS - Blurred vision; Eye pain; Photophobia; 
Vision decreased 
GASTROINTESTINAL DISORDERS - Abdominal 
distension; Colitis; Dysphagia; Esophagitis; Gastritis; 

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Gastroesophageal reflux disease; Gastrointestinal disorders - 
Other (intestinal obstruction); Gastrointestinal disorders - 
Other (intestinal perforation);  Gastrointestinal disorders - 
Other (salivary hypersecretion); Hemorrhoids; Ileus; Oral 
pain; Pancreatitis; Small intestinal obstruction; Small 
intestinal perforation; Toothache 
GENERAL DISORDERS AND ADMINISTRATION SITE 
CONDITIONS - Chills; Flu like symptoms; Gait disturbance; 
General disorders and administration site conditions - Other 
(groin pain); Malaise; Non-cardiac chest pain; Pain 
HEPATOBILIARY DISORDERS - Gallbladder obstruction 
IMMUNE SYSTEM DISORDERS - Allergic reaction 
INFECTIONS AND INFESTATIONS - Abdominal infection; 
Infections and infestations - Other (cystitis); Infections and 
infestations - Other (lower respiratory tract infection); 
Infections and infestations - Other (mucosal infection); 
Infections and infestations - Other (parotid gland); Kidney 
infection; Lung infection; Papulopustular rash; Sepsis; Skin 
infection; Upper respiratory infection 
INJURY, POISONING AND PROCEDURAL 
COMPLICATIONS - Fall; Fracture; Injury, poisoning and 
procedural complications - Other (accidental overdose); 
Injury, poisoning and procedural complications - Other 
(postoperative fever); Injury, poisoning and procedural 
complications - Other (subdural hemorrhage); Tracheal 
obstruction 
INVESTIGATIONS - Alanine aminotransferase increased; 
Alkaline phosphatase increased; Aspartate aminotransferase 
increased; Blood bilirubin increased; Blood lactate 
dehydrogenase increased; Cholesterol high; GGT increased; 
Lipase increased; Lymphocyte count decreased; Neutrophil 
count decreased; White blood cell decreased 
METABOLISM AND NUTRITION DISORDERS - Acidosis; 
Hypercalcemia; Hyperkalemia; Hypernatremia; 
Hypertriglyceridemia; Hyperuricemia; Hypoalbuminemia; 
Hypocalcemia; Hyponatremia; Metabolism and nutrition 
disorders - Other (severe chronic malnutrition); Metabolism 
and nutrition disorders - Other (vitamin D deficiency)  
MUSCULOSKELETAL AND CONNECTIVE TISSUE 
DISORDERS - Bone pain; Chest wall pain; Flank pain; 
Generalized muscle weakness; Muscle cramp; Myalgia 
NEOPLASMS BENIGN, MALIGNANT AND 
UNSPECIFIED (INCL CYSTS AND POLYPS) - Neoplasms 
benign, malignant and unspecified (incl cysts and polyps) - 
Other (non-hodgkin lymphoma); Treatment related 

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secondary malignancy 
NERVOUS SYSTEM DISORDERS - Ataxia; Intracranial 
hemorrhage; Lethargy; Nervous system disorders - Other 
(carotid artery occlusion); Nervous system disorders - Other 
(neuropathy peripheral); Paresthesia; Radiculitis; Stroke; 
Tremor 
PSYCHIATRIC DISORDERS - Anxiety; Confusion; 
Depression; Psychiatric disorders - Other (mental status 
changes) 
RENAL AND URINARY DISORDERS - Dysuria; 
Hematuria; Proteinuria; Renal and urinary disorders - Other 
(strangury) 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - 
Vaginal hemorrhage 
RESPIRATORY, THORACIC AND MEDIASTINAL 
DISORDERS - Bronchopulmonary hemorrhage; Epistaxis; 
Hiccups; Hypoxia; Nasal congestion; Pleural effusion; 
Pleuritic pain; Pneumothorax; Postnasal drip; Productive 
cough 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - 
Alopecia; Dry skin; Hyperhidrosis; Rash acneiform; 
Urticaria 
VASCULAR DISORDERS - Flushing; Hypertension; 
Hypotension; Thromboembolic event 
Note: MLN0128 (TAK-228) in combination with other agents 
could cause an exacerbation of any adverse event currently 
known to be caused by the other agent, or the combination 
may result in events never previously associated with either 
agent. 
4. 13.1 Study 
Design/ 
Endpoints 
89 
PI RESPONSE: The statistical collaborator was updated. 
Old Text: 
Dr. Kenneth Hess (Professor of Biostatistics at UT MD Anderson 
Cancer Center) will perform all statistical analyses. 
New Text:  
Dr. Suyu Liu (Biostatistics at UT MD Anderson Cancer Center) 
will perform all statistical analyses. 
 
 
 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
1 
 
NCI Protocol #: 
9585  
 
Local Protocol #: 
2013-0665 
 
TITLE: PHASE I STUDY OF MLN0128 (TAK-228) (NSC# 768435) IN 
COMBINATION WITH ZIV-AFLIBERCEPT (NSC# 724770) IN PATIENTS WITH 
ADVANCED CANCERS 
 
Coordinating Center: 
The University of Texas, MD Anderson Cancer Center 
 
*Principal Investigator: 
Aung Naing 
 
1515 Holcombe Blvd, Unit 455 
 
Houston, Texas 77030 
 
Phone: 713-563-0803 
 
Fax: 713-563-0566 
 
Email: anaing@mdanderson.org 
 
Co-Investigators: 
 
Funda Meric-Bernstam, MD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-745-4453 
Fax: 713-404-3176 
Email: fmeric@mdanderson.org 
 
Siqing Fu, MD, PHD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-792-7960 
Email: siqingfu@mdanderson.org 
 
David S. Hong, MD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-563-5844 
Email: dshong@mdanderson.org 
 
Filip Janku, MD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-563-2632 
Email: FJanku@mdanderson.org 
 
Daniel Karp, MD 
1515 Holcombe Blvd., Unit 455 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
2 
 
Houston, TX 77030 
Phone: 713-745-7398 
Email: dkarp@mdanderson.org 
 
Sarina Piha-Paul, MD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-794-4225 
Email: spihapau@mdanderson.org 
 
Vivek Subbiah, MD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-563-0393 
Email: vsubbiah@mdanderson.org 
 
Apostolia Tsimberidou, MD, PHD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-792-4259 
Email: atsimber@mdanderson.org 
 
Shubham Pant, MD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-745-6601 
Email: spant@mdanderson.org 
 
Jordi Rodon Ahnert, MD, PHD 
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-792-5603 
Email: jrodon@mdanderson.org  
 
Timothy A. Yap, MD, PHD  
1515 Holcombe Blvd., Unit 455 
Houston, TX 77030 
Phone: 713-563-1784 
Email: tyap@mdanderson.org 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
3 
 
Collaborators 
Shalini Dalal, MD* 
Jean-Bernard Durand, MD* 
Palliative Care 
Cardiology 
1515 Holcombe Blvd., Unit 1414 
1515 Holcombe Blvd., Unit 1451 
Houston, TX 77030 
Houston, TX 77030 
Phone: 713-792-6085 
Phone: 713-792-6239 
Email: sdalal@mdanderson.org 
Email: jdurand@mdanderson.org 
 
Mouhammed A. Habra, MD* 
Ravi Murthy, MD* 
Endocrine Neoplasia and HD 
Interventional Radiology 
1515 Holcombe Blvd., Unit 1461 
1515 Holcombe Blvd., Unit 1471 
Houston, TX 77030 
Houston, TX 77030 
Phone: 713-792-2841 
Phone: 713-745-0856 
Email: mahabra@mdanderson.org 
Email: rmurthy@mdanderson.org 
 
Chaan Ng, MD* 
Sinchita Roy Chowdhuri, MD, PHD* 
Diagnostic Radiology 
Pathology 
1515 Holcombe Blvd., Unit 1473 
1515 Holcombe Blvd., Unit 0085 
Houston, TX 77030 
Houston, TX 77030 
Phone: 713-792-6759 
Phone: 713-563-9344 
Email: cng@mdanderson.org 
Email: sroy2@mdanderson.org 
 
Ana M. Ciurea, MD* 
Nizar M. Tannir, MD 
Dermatology 
Genitourinary Medical Oncology 
1515 Holcombe Blvd., Unit 0434 
1515 Holcombe Blvd., Unit 1374 
Houston, TX 77030 
Houston, TX 77030 
Phone: 713-563-6645 
Phone: 713-563-7265 
Email: amciurea@mdanderson.org 
Email: ntannir@mdanderson.org 
 
Peter Kim, MD* 
Ala Abudayyeh, MD* 
Cardiology 
Nephrology 
1515 Holcombe Blvd., Unit 1451 
1515 Holcombe Blvd., Unit 1468 
Houston, TX 77030 
Houston, TX 77030 
Phone: 713-792-6246 
Phone: 713-404-9366 
Email: pkim@mdanderson.org 
Email: aabudayyeh@mdanderson.org 
 
 
* Collaborators who will not be consenting or treating patients on trial. 
 
Statistician:  
 
 
 
 
Study Coordinator: 
Suyu Liu, PhD 
Fengying “Fiona” Ouyang 
1515 Holcombe Blvd., Unit 1409 
1515 Holcombe Blvd., Unit 455 
Houston, Texas 77030 
Houston, Texas 77030 
713-563-4280 
713-792-7192 
syliu@mdanderson.org 
713-404-5990 
fouyang@mdanderson.org 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
4 
 
 
Responsible Research Nurse: 
 
 
Responsible Data Manager: 
Jing Gong 
Senait Fessahaye 
1515 Holcombe Blvd., Unit 455 
1515 Holcombe Blvd., Unit 455 
Houston, Texas 77030 
Houston, Texas 77030 
713-563-8392 
713-563-9897 
713-606-3681 
713-792-6402 
jinggong@mdanderson.org 
snfessah@mdanderson.org 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
5 
 
 
NCI-Supplied Agent(s): MLN0128 (TAK-228) (NSC# 768435), Millennium 
Pharmaceuticals, Inc. IND #: 121931 
IND Sponsor: DCTD, NCI 
 
Additional NCI-Supplied Agent(s):  Ziv-Aflibercept (NSC# 724770), Sanofi Aventis 
Corporation 
IND #: 100137 
IND Sponsor: DCTD, NCI 
 
Protocol Type / Version # / Version Date  
Original/ Local Version #00 / February 24, 2014 
Amendment 01/ Local Version #01 / March 28, 2014 
Amendment 02/ Local Version #02 / August 15, 2014 
Amendment 03/ Local Version #03 / October 21, 2014 
Amendment 04/ Local Version #03 / November 7, 2014 
Amendment 05/ Local Version #03 / February 2, 2015 
Amendment 06/ Local Version #03 / March 20, 2015 
Amendment 07/ Local Version #04 / October 6, 2015  
Amendment 08/ Local Version #05/ January 5, 2016 
Amendment 09/ Local Version #06/ November 2, 2016 
Amendment 10/ Local Version #07/ August 3, 2017 
Amendment 11/ Local Version #07/ September 21, 2017 
Amendment 12/ Local Version #07/ December 22, 2017 
Amendment 13/ Local Version #07/ January 29, 2018 
Amendment 14/ Local Version #08/ June 20, 2018 
Amendment 15/ Local Version #09/ June 11, 2019 
Amendment 16/ Local Version #10/ October 8, 2019 
 
 
 
Disclaimer Statement: 
This is a research protocol of the University of Texas M. D. Anderson Cancer Center describing an experimental treatment 
procedure. It is a privileged document and is not intended to be circulated, copied or used for other purposes without the 
expressed written consent of the Principal Investigator.  The University of Texas M. D. Anderson Cancer Center assumes no 
responsibility for it use outside of the constraints of this research protocol or by investigators other than those listed on this 
document. 

NCI Protocol #: 9585 
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SCHEMA 
 

NCI Protocol #: 9585 
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7 
 
 
Dose Escalation Schedule 
Dose Level 
Dose* 
MLN0128 (TAK-228) 
(mg PO for 3 days on and 4 days 
off) Starting on Cycle 1 Day 2 
Ziv-Aflibercept (mg/kg IV Q 
2 weeks) 
Starting on Cycle 1 Day 1 
Level -1 
3 
2 
Level 1 
4 
2 
Level 2 
4 
3 
*Doses are stated as exact dose in units (e.g., mg/m2, mcg/kg, etc.) rather than as a percentage. 
As of Amendment 13/ Version 07, all newly enrolled patients will be given the milled formulation of MLN0128 
(TAK-228), and a new dose escalation is being explored. Starting with Amendment 14/Version 08, the dose 
escalation will be re-started at Dose Level 1 with the updated dose level escalation. 
Both study drugs will have a +/- 2 day window. If the ziv-aflibercept dosing is rescheduled, then the MLN0128 
(TAK-228) dosing will be adjusted accordingly. 
 
Dose Escalation Treatment Schema 
 
 
 
 
In cases where a subject misses dosing at his/her dosing time, the subject may still take the 
dose within 12 hours of the regular dosing time (subjects should not take 2 consecutive daily 
doses within 12 hours of each other).  Subjects who vomit after receiving MLN0128 (TAK-
228) will not receive a replacement dose within 24 hours.  If confirmed that the study drug 
has been vomited, the dose should be noted as having been missed. 
 
 
Dose Expansion Treatment and Correlative Study Schema 

NCI Protocol #: 9585 
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9 
 
TABLE OF CONTENTS 
TITLE: PHASE I STUDY OF MLN0128 (TAK-228) (NSC# 768435) IN COMBINATION 
WITH ZIV-AFLIBERCEPT (NSC# 724770) IN PATIENTS WITH ADVANCED 
CANCERS ........................................................................................................................ 1 
SCHEMA ...................................................................................................................................... 6 
Table of Contents .......................................................................................................................... 9 
1. 
OBJECTIVES ................................................................................................................. 11 
1.1 Primary Objectives .................................................................................................... 11 
1.2 
Secondary Objectives .......................................................................................... 11 
2. 
BACKGROUND ............................................................................................................ 11 
2.1 
Study Disease(s).................................................................................................. 11 
2.2 
MLN0128 (TAK-228) (INK128) ........................................................................ 11 
2.3 
VEGF-Trap (Ziv-Aflibercept) ............................................................................. 23 
2.4 
Rationale for Combination of MLN0128 (TAK-228) and VEGF-Trap (Ziv-
Aflibercept) ......................................................................................................... 28 
2.5 
Correlative Studies Background .......................................................................... 29 
3. 
PATIENT SELECTION ................................................................................................. 31 
3.1 
Inclusion Criteria ................................................................................................. 31 
3.2 
Exclusion Criteria ................................................................................................ 32 
3.3 
Inclusion of Women and Minorities .................................................................... 34 
4. 
REGISTRATION PROCEDURES ................................................................................ 35 
4.1 
General Guidelines .............................................................................................. 35 
4.2 
Registration Process ............................................................................................ 35 
5. 
TREATMENT PLAN ..................................................................................................... 35 
5.1 
Agent Administration .......................................................................................... 35 
5.2 
Definition of Dose-Limiting Toxicity ................................................................. 38 
5.3 
General Concomitant Medication and Supportive Care Guidelines .................... 40 
5.4 
Duration of Therapy ............................................................................................ 44 
5.5 
Duration of Follow Up ........................................................................................ 44 
5.6 
Criteria for Removal from Study......................................................................... 44 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ............................................................. 45 
7. 
ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS .......................... 54 
7.1 Comprehensive Adverse Events and Potential Risks List(s) (CAEPRs) .................. 54 
7.2 
Adverse Event Characteristics ............................................................................. 62 
7.3 
Expedited Adverse Event Reporting ................................................................... 62 
7.4 
Routine Adverse Event Reporting ....................................................................... 64 
7.5 
Secondary Malignancy ........................................................................................ 64 
7.6 
Second Malignancy ............................................................................................. 64 

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8. 
PHARMACEUTICAL INFORMATION ....................................................................... 65 
8.1 
CTEP IND Agent(s) ............................................................................................ 65 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ..................................... 69 
9.1 
Immunohistochemistry (IHC) ............................................................................. 70 
9.2 
Reverse Phase Protein Assay (RPPA) ................................................................ 71 
10. 
STUDY CALENDAR .................................................................................................... 74 
11. 
MEASUREMENT OF EFFECT..................................................................................... 77 
11.1 
Antitumor Effect – Solid Tumors ....................................................................... 77 
11.2 
Antitumor Effect – Hematologic Tumors ........................................................... 83 
11.3 
Other Response Parameters ................................................................................. 83 
12. 
DATA REPORTING / REGULATORY REQUIREMENTS ........................................ 83 
12.1 
Data Reporting .................................................................................................... 83 
12.2 
CTEP Multicenter Guidelines ............................................................................. 84 
12.3 
Collaborative Agreements Language .................................................................. 84 
13. 
STATISTICAL CONSIDERATIONS............................................................................ 86 
13.1 
Study Design/Endpoints...................................................................................... 86 
13.2 
Sample Size/Accrual Rate ................................................................................... 87 
13.3 
Stratification Factors ........................................................................................... 88 
13.4 
Analysis of Secondary Endpoints ....................................................................... 88 
REFERENCES ........................................................................................................................... 90 
APPENDIX A PERFORMANCE STATUS CRITERIA ........................................................ 93 
APPENDIX B 
NEW YORK HEART ASSOCIATION (NYHA) CLASSIFICATIONS ....... 94 
APPENDIX C INFORMATION ON POSSIBLE DRUG INTERACTIONS......................... 95 
APPENDIX D PATIENT’S MEDICATION DIARY - AGENT MLN0128 (TAK-228) ..... 101 
APPENDIX E 
GLUCOSE MONITORING INSTRUCTIONS ............................................ 104 
APPENDIX F 
BIOMARKER ASSAY CHARACTERIZATION ........................................ 105 
APPENDIX G COLLECTION AND PROCESSING OF TUMOR BIOPSY AND SAMPLES
 
122 
APPENDIX H PROCEDURES FOR ISOLATING PERIPHERAL BLOOD 
MONONUCLEAR CELLS (PBMC) AND PLASMA ................................................ 125 
APPENDIX I 
IMMUNOHISTOCHEMICAL (IHC) MARKER TEMPLATE FOR 
INTEGRAL MARKERS IN CLINICAL TRIALS ...................................................... 128 

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1. OBJECTIVES 
 
1.1 Primary Objectives 
 
1.1.1 To evaluate safety and tolerability, determine maximum tolerated dose 
(MTD) and recommend a phase II dose of the combination of 
MLN0128 (TAK-228) with ziv-aflibercept in patients with advanced 
cancers refractory to standard therapy. 
 
1.2 
Secondary Objectives 
 
1.2.1 To give early indication of efficacy by evaluation of tumor size. 
 
1.2.2 To evaluate Akt/mTOR signaling and adaptive responses; testing 
phosphorylation levels of biomarkers such as, but not limited to, 
VEGF1 and 2, AKT and 4E-BP1 following treatment with MLN0128 
(TAK-228) and ziv-aflibercept in PBMCs and biopsy samples during 
expansion cohort. 
 
 
2. BACKGROUND 
 
2.1 
Study Disease(s) 
 
PI3K/AKT/mTOR Pathway and Cancer 
 
The PI3K/AKT/mTOR pathway is one of the most frequently deregulated pathways in human 
tumors, although mTOR mutations have not been reported. Pathway activation occurs either by 
mutation or upregulation of upstream proteins (such as PI3K or AKT) or through loss of PTEN 
(mutation or epigenetic silencing). Enhanced growth factor receptor signaling via amplification, 
mutation or autocrine stimulation, can also lead to pathway activation. Finally, amplification of 
BCL2, constitutive activation of the Ras/RAF/MEK pathway, c-Abl kinase and cyclin D1 
overexpression have been shown to activate the PI3K/AKT/mTOR pathway and are present in 
variable amounts in solid tumors and hematological malignancies. Around 70% of late-stage 
cancer patients have specific activation patterns in at least one of these pathways and blocking 
these effects at the point of convergence is a rational approach in cancer therapy (Meric-  
Bernstam and Mills, 2004). Deregulation of the pathway is characterized by high mTORC1 and 
mTORC2 activity, associated with high pS6K and pAKT levels. This is particularly prominent in 
solid tumors from lung, breast, head and neck, ovary and colon (Dancey, 2004; Georgakis and  
Younes, 2006; Fasolo and Sessa, 2008). 
 
2.2 
MLN0128 (TAK-228) (INK128) 
 
MLN0128 (TAK-228) is an orally bioavailable, potent, highly selective, adenosine 5’ 
triphosphate (ATP)-competitive inhibitor of the kinase activity of mammalian target of 

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rapamycin (mTOR) (Chiang et al., 2007). mTOR is a serine/threonine kinase belonging to the 
phosphatidylinositol 3 kinase (PI3K)-related kinase superfamily. It functions as a sensor of 
mitogen activity, cellular energy and nutrient levels, and it is a central controller of cell growth. 
 
Mechanism of Action 
The mTOR is a kinase that regulates cell growth, translational control, angiogenesis, and cell 
survival by integrating nutrient and hormonal signals.  mTOR kinase plays a key role in several 
pathways that are frequently dysregulated in human cancer (Chiang et al.,  2007).  mTOR exists 
in two complexes, one with raptor, which is rapamycin-sensitive, and the other with rictor, 
which is rapamycin insensitive. mTOR complex 1 (mTORC1) phosphorylates 4EPB1 and 
p70S6 kinase and results in translation of proteins involved in cell cycle progression. The 
mTOR complex 2, rapamycin-insensitive (mTORC2), has been shown to directly phosphorylate 
and activate the upstream kinase AKT at serine 473. Inhibition of mTORC1 inhibits the negative 
feedback loop between S6 kinase and insulin receptor substrate 1 (Figure 1) (Naing, 2013). This 
results in an increase in PI3K and AKT activity and may explain, at least in part, the limited 
activity of rapamycin analogs in a number of tumor types. 
 
Inhibiting mTOR may inhibit abnormal cell proliferation, tumor angiogenesis, and abnormal 
cellular metabolism, thus providing the rationale for mTOR inhibitors as potential agents as 
either monotherapy or in combination with other chemotherapeutic agents in the treatment of 
solid tumor and hematological malignancies. MLN0128 (TAK-228) inhibits both mTORC1 and 
mTORC2 complexes by inhibiting mTOR kinase. It, therefore, controls both S6K and AKT; and 
is expected to have a broader spectrum of activity than rapamycin and its analogs in man. 
 
Figure 1: Inhibition of mTORC1 and mTORC2 by dual kinase inhibitor (Modified figure 
from Naing, 2013) 
 
To address the incomplete inhibition of mTOR by rapalogs, MLN0128 (TAK-228) was 
developed. MLN0128 (TAK-228) is a potent, selective, orally bioavailable, ATP-competitive 
inhibitor of mTOR that is currently in phase 1 clinical trials. MLN0128 (TAK-228) targets both 

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mTORC1 and TORC2, and is designed to overcome the shortcomings of the current rapalogs to 
achieve greater clinical benefit (Investigator’s Brochure, 2017). 
 
Nonclinical Studies with MLN0128 (TAK-228) in Human Tumor Models 
 
MLN0128 (TAK-228) selectively and potently inhibits mTOR kinase (IC50 = 1.1 nM), inhibits 
mTORC1/2 signaling, and prevents cellular proliferation. The in vitro cellular potency of 
MLN0128 (TAK-228) was not diminished in the presence of human whole blood components.  
 
MLN0128 (TAK-228) displayed cellular inhibition of TORC1 and TORC2 pathways with IC50 
less than 10 nM. MLN0128 (TAK-228) is believed to have the potential of achieving greater 
clinical benefit than the currently available rapalogs. In vitro studies have demonstrated that 
MLN0128 (TAK-228) MLN0128 selectively and potently inhabits the mTOR kinases, but 
relative to mTOR inhibition MLN0128 (TAK-228) has >100-fold less potency as an inhibitor of 
Class I (PI3 kinase isoforms α, β, γ, δ), class II (PI3KC2α and PI3K2C β), and class III (VPS34) 
PI3K family members, as well as PI3Kα and PI3Kβ. (Investigator’s Brochure, 2017).  MLN0128 
(TAK-228) was also found to inhibit (>80%) of the biochemical activity of only five kinases 
(mTOR, DNA-PK, PDGFR α, Flt3, and CK1 epsilon kinases) out of a panel of 222 protein 
kinases. MLN0128 (TAK-228) inhibited ligand binding of 10 receptor and intracellular protein 
kinases including (ACVR1, BMPR1B, CSF1R, CSNK1D, CSNK1E, DDR1, MEK1, MEK2, 
PDGFR α, and RIPK2) out of a panel of 402 distinct kinases. In PTEN deficient cells, MLN0128 
(TAK-228) had pharmacodynamic properties comparable to dual inhibition of TORC1 and PI3K 
utilizing a combination of rapamycin and PI-103. 
 
The pharmacodynamics and antitumor activity of MLN0128 (TAK-228) was studied in vivo in 
murine xenograft models of human glioblastoma, NSCLC, breast cancer, renal cell cancer, 
endometrial adenocarcinoma, and castration-resistant prostate cancer (CPRC). Consistent with 
the mode of action, MLN0128 (TAK-228) inhibited phosphorylation of downstream modulators 
of mTORC1 (namely 4EBP1 and S6) and mTORC2 (namely AKT [S473]) in human U87 
glioblastoma tumor xenograft models in mice at doses as low as 0.1 mg/kg. Additionally, 
MLN0128 (TAK-228) showed strong tumor growth inhibition (TGI) in all 8 xenograft models at 
tolerable oral (PO) doses from 0.15 mg/kg (daily [QD]; tested in MDA-MB-361 breast 
carcinoma) to 3.0 mg/kg (every other day [Q2D] or once weekly [QW]; tested in all models) 
(Investigator’s Brochure, 2017). 
 
In addition to single-agent activity in these xenograft models, MLN0128 (TAK-228) was 
combined with the standard of care (SOC) agent paclitaxel in the breast and endometrial 
models. The combination of MLN0128 (TAK-228) with paclitaxel resulted in enhanced 
antitumor activity and reduced tumor burden. When tumors were pretreated with paclitaxel, 
there was an added benefit in tumor reduction compared to the effects of simultaneous 
administration of both agents, consistent with the mechanism of action of paclitaxel. Findings 
from these nonclinical pharmacology studies suggest that MLN0128 (TAK-228), alone and in 
combination with paclitaxel, has therapeutic potential as an orally administered mTORC1/2 
inhibitor for the treatment of cancers associated with dysregulated activation of the 
PI3K/AKT/mTOR pathway, such as renal cell, endometrial, breast, lung, and prostate 
cancers(Investigator’s Brochure, 2017). 
 
 

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In preliminary work, we evaluated the antitumor efficacy of MLN0128 (TAK-228) in 
intrinsically rapamycin-resistant and rapamycin-sensitive cancer cell lines (Figure 2). We 
demonstrated that both rapamycin-sensitive and resistant cancer cell lines were responsive to 
clinically achievable levels of MLN0128 (TAK-228) (Figure 2A). Evaluation of the effect of 
MLN0128 (TAK-228) on downstream signaling in multiple cell lines have shown more robust 
inhibition of mTOR targets S6K and 4E-BP1, as well as inhibition of phospho-Akt Ser473. 
There was, however, only transient inhibition of phospho-Akt Thr 308, with subsequent 
upregulation (Figure 2B).  Further, in some in vivo models MLN0128 (TAK-228) had a 
significantly greater antitumor efficacy compared to rapamycin (unpublished data). 
 
 
Figure 2. In vitro efficacy of MLN0128 (TAK-228). A. A panel of cancer cell lines 
with differential sensitivity to rapamycin was treated with increasing doses of MLN 
0128 and rapamycin; Half maximal inhibitory concentrations (IC50s) were determined 
by Sulforhodamine B colorimetric (SRB) assay. B. Cell lines were treated with 
vehicle, rapamycin or MLN0128 (TAK-228) for 24 hours and cell signaling was 
assessed by Western blotting. Results from 2 of 13 cell lines evaluated are shown. 
 
MLN0128 (TAK-228) Nonclinical Toxicology and PK studies 
 
PK Studies 
MLN0128 (TAK-228) was rapidly absorbed after oral administration to mice, rats, dogs, and 
monkeys, with high oral bioavailability.  MLN0128 (TAK-228) did not inhibit P-glycoprotein (P- 
gp).  A study of the tissue distribution of [14C] MLN0128 (TAK-228) showed that [14C] 
MLN0128 (TAK-228) was rapidly and widely distributed throughout the body in Long-Evans 
rats; radioactivity was eliminated from most tissues at 48 hours post dose, and from all but the 

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adrenal cortex, adrenal gland, adrenal medulla, eye, liver, and uveal tract at 168 hours.  
MLN0128 (TAK-228) displayed dose proportional plasma exposures and a moderate propensity 
to cross the blood-brain barrier.  MLN0128 (TAK-228) was modestly bound to human plasma 
proteins (approximately 70%).  MLN0128 (TAK-228) inhibited breast cancer resistance protein 
(BCRP), organic cation transporter (OCT)1, and OCT2 (Investigator’s Brochure, 2017). 
 
M1, the single metabolite (monohydroxylation product) observed in human microsomal 
incubations, was also observed in rats and monkeys, the species used for the Good Laboratory 
Practice (GLP) toxicology studies. Recently completed in vitro metabolism experiments in 
human hepatocytes using 14C-labeled TAK-228 suggest that TAK-228 is metabolized primarily 
via CYP1A2 (approximately 31%-40%), with a minor contribution from CYP3A4 
(approximately 11%-22%). These data suggest that TAK-228 is also metabolized by direct 
glucuronidation (approximately 22%) and an unidentified non-uridine diphosphate glucuronosyl 
transferase pathway (approximately 18%). The new data differ from the previous in vitro CYP 
phenotyping data obtained using recombinant CYP enzymes, which suggested the involvement 
of CYP2C9 (approximately 35%), CYP2C19 (approximately 28%), and CYP3A4 
(approximately 28%) in TAK-228 metabolism. In addition, physiologically based PK modeling 
and simulation using the new metabolism data for TAK-228 suggest that the risk for a 
metabolism-based drug-drug interaction with TAK-228 appears to be low. 
 
 
Toxicology 
The MLN0128 (TAK-228) toxicology program consisted of single- and repeat-dose studies in 
rats and monkeys, single-dose studies in dogs, and an Ames genotoxicity study. The toxicologic 
profiles obtained in the non-GLP-compliant and GLP-compliant studies were generally 
consistent. The observed toxicities were consistent between rats and monkeys, with no apparent 
sex differences (Investigator’s Brochure, 2017). 
 
The toxicity profile of MLN0128 (TAK-228) in rats and monkeys, as established in GLP- 
compliant repeat-dose studies, is consistent with pharmacologic inhibition of mTORC1/2 
activity.  The dose limiting toxicities (DLTs) of MLN0128 (TAK-228) in rats and monkeys were 
secondary to an exaggerated pharmacologic response and consisted of body weight loss and 
associated clinical observations that included gastrointestinal (GI) distress and decreased 
activity, appetite, and body temperature. Adverse effects in rats included body weight loss, 
decreased activity, increased glucose and insulin levels, alterations in white blood cells (WBCs), 
bone marrow and lymphoid depletion, thymic necrosis, oligospermia, testes degeneration/ 
atrophy, nonglandular stomach epithelial degeneration/ ulceration/ hyperplasia, and alveolar 
histiocytosis. The microscopic findings observed in the testes, epididymides, and nonglandular 
stomach were not resolved after a 14-day recovery period, while partial to complete resolution 
was seen in the lungs, thymus, and bone marrow.  The adverse effects in monkeys included 
decreased activity, appetite, and body weight; increased glucose and insulin; lymphoid and bone 
marrow depletion; adrenal hypertrophy/hyperplasia; pancreatic and salivary gland acinar cell 
secretory depletion; GI tract erosion and ulceration; and skin ulceration/epidermal hyperplasia. 
The findings in the pancreas, adrenal glands, and salivary glands may have been related to a 
stress response or reduced food consumption. The findings were generally reversible after a 14-
day recovery period (Investigator’s Brochure, 2017). 

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The findings in rat and monkey repeat-dose toxicology studies with MLN0128 (TAK-228), 
including bone marrow and lymphoid depletion, GI and skin effects, and effects on glucose and 
insulin levels, can be monitored in clinical trials. The toxicities seen in the repeat-dose 
toxicology studies, such as GI effects and glucose and insulin increases, are consistent with the 
treatment-emergent adverse events (TEAEs), including mucositis and hyperglycemia, observed 
to date in patients receiving MLN0128 (TAK-228).  MLN0128 (TAK-228) was negative for 
mutagenicity in the Ames assay, and shows low potential for phototoxicity (Investigator’s 
Brochure, 2017).  MLN0128 (TAK-228) has a low potential to affect the hERG potassium ion 
channel, and did not affect cardiovascular parameters in vivo in telemeterized monkeys 
(Investigator’s  Brochure, 2017). 
 
Summary of MLN0128 (TAK-228) Clinical Studies 
 
Single-agent MLN0128 (TAK-228) had initial clinical development in two phase 1 studies in 
subjects with advanced solid malignancies and hematologic malignancies (multiple myeloma 
[MM] and Waldenstrom macroglobulinemia [WM]), and in a third study in combination with 
paclitaxel with or without trastuzumab in subjects with advanced solid tumors (Investigator’s 
Brochure, 2017). 
 
Study INK128-001 
Study INK128-001 evaluated safety and anti-tumor activity of MLN0128 (TAK-228) in 
subjects with advanced solid malignancies. As of 13 September 2016, 198 subjects have 
been treated in Study INK128-001.  As of the cutoff date, treatment-emergent SAEs had 
been reported for 84 patients (42%) in this study. The most commonly reported (≥4 
patients, overall) preferred terms were stomatitis in 7 patients (4%), pneumonia in 6 
patients (3%), abdominal pain, acute kidney injury, or anemia in 5 each (3%), and 
asthenia or vomiting in 4 each (2%). 
 
All 116 patients in the dose escalation phase reported at least 1 treatment-emergent 
adverse event (TEAE). Across all dosing schedules in this phase, the most frequently 
occurring TEAEs were hyperglycemia, nausea, and vomiting. There were no meaningful 
trends across dosing schedules or dose levels in the incidence of these TEAEs. 
 
The most commonly reported TEAEs among patients receiving QD dosing (n=31) were 
hyperglycemia (84%) and nausea (52%). The incidence of hyperglycemia increased in a 
dose-dependent manner. A total of 19 QD patients (61%) overall reported at least 1 
TEAE of Grade 3 or greater severity. The most commonly reported TEAEs of Grade 3 or 
greater severity were hyperglycemia (6 patients [19%] overall) and lymphopenia (3 
patients [10%] overall). 
 
Among patients receiving QW dosing (n=30), the most commonly reported TEAEs were 
nausea (73%), vomiting (67%), and hyperglycemia (60%). Clear dose relationships were 
not evident among the most commonly reported TEAEs, but the most common TEAEs 
were generally reported more frequently by patients in the 30 and 40 mg than patients in 
the 7 and 10 mg QW dose groups. A total of 18 patients (60%) overall reported at least 1 
TEAE of Grade 3 or greater severity. The most common TEAE of Grade 3 or greater 

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severity was fatigue (3 patients [10%] overall). TEAEs of Grade 3 or greater severity 
were reported more frequently by patients in the 40 mg than patients in the other QW 
dose groups. 
 
Among patients receiving QD×3d QW dosing (n=33), the most commonly reported 
preferred terms were nausea (73%), stomatitis (70%), hyperglycemia (67%), decreased 
appetite and vomiting (61% each), and diarrhea (52%). Clear dose relationships were not 
evident among the most commonly reported TEAEs. A total of 25 patients (76%) overall 
reported at least 1 TEAE of Grade 3 or greater severity. The most common (≥10% of 
patients) TEAEs of Grade 3 or greater severity were hyperglycemia (7 patients [21%] 
overall) and asthenia, hypophosphatemia, and stomatitis (each reported by 5 patients 
[15%] overall). TEAEs of Grade 3 or greater severity were reported less frequently by 
patients in the 6 and 9 mg QD×3d QW than patients in the other dose groups. 
 
The most commonly reported TEAEs among patients receiving QD×5d QW dosing 
(n=22) were nausea and stomatitis (59% each) and hyperglycemia and vomiting (55% 
each). Events of stomatitis, vomiting, and decreased appetite increased in a dose-
dependent manner across the dose levels in the QD×5d QW schedule. A total of 14 
patients (64%) overall reported at least 1 TEAE of Grade 3 or greater severity. The most 
common (≥10% of patients) TEAEs of Grade 3 or greater severity were asthenia and 
stomatitis (each reported by 3 patients [14%] overall). TEAEs of Grade 3 or greater 
severity were reported more frequently by patients in the 13 mg than patients in the 7 and 
10 mg QD×5d QW dose groups. 
 
All 82 patients in the expansion phase reported at least 1 TEAE. Across dosing schedules 
in this phase, the most frequently occurring TEAEs were fatigue, nausea, and 
hyperglycemia. There were no meaningful differences between dosing schedules in the 
overall incidence of these TEAEs or Grade 3 or greater TEAEs, and the pattern of these 
events was generally similar in both schedules. 
 
Among patients receiving 5 mg QD dosing during the Expansion Phase (n=39), the most 
commonly reported (>50% of patients) preferred terms were fatigue (64%), nausea 
(54%), and decreased appetite and diarrhea (51% each). A total of 30 patients (77%) in 
the 5 mg QD dose group reported at least 1 TEAE of Grade 3 or greater severity. The 
most common (≥10% of patients) TEAEs of Grade 3 or greater severity were 
hyperglycemia and fatigue (each reported by 5 patients [13%]) and hypophosphatemia (4 
patients [10%]). 
 
The most commonly reported TEAEs among patients receiving 30 mg QW dosing (n=17) 
were nausea (76%), hyperglycemia (71%), fatigue (65%), vomiting (59%), and stomatitis 
(53%). A total of 9 patients (53%) in the 30 mg QW dose group reported at least 1 TEAE 
of Grade 3 or greater severity. The most common (≥10% of patients) TEAEs of Grade 3 
or greater severity were hyperglycemia, fatigue, hypophosphatemia, nausea, and pain in 
extremity (each reported by 2 patients [12%]). 
 
Among patients receiving 40 mg QW dosing (n=26), the most commonly reported (>50% 

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of patients) preferred terms were fatigue (92%), nausea, hyperglycemia, and vomiting 
(77% each), and decreased appetite (54%). A total of 19 patients (73%) in the 40 mg QW 
dose group reported at least 1 TEAE of Grade 3 or greater severity. The most common 
(≥10% of patients) TEAEs of Grade 3 or greater severity were hyperglycemia (6 patients 
[23%]), fatigue (4 patients [15%]), and anemia and dehydration (each reported by 3 
patients [12%]). 
 
In Study INK128-001, as of 13 September 2016, the maximum tolerated doses (MTD) 
for all 4 schedules had been determined: for the QD dosing the MTD is 6 mg QD, for the 
QDx3dQW dosing, the MTD is 16 mg; for the QDx5dQW dosing, the MTD is 10 mg; 
and for the QW dosing schedule, the MTD is 40 mg. The MTDs for each of the 4 
schedules was determined by evaluation of cohorts of 6 evaluable patients. At each MTD, 
up to 6 additional patients were enrolled to further evaluate safety and tolerability. A 
significant proportion of patients treated at the MTDs required dose modifications due to 
drug-related AEs beyond 1 or 2 cycles, and therefore were not representative of a 
recommended phase 2 dose. The study is currently further evaluating doses at less than 
the MTD for QDx3dQW and QDx5dQW, to determine a dose(s) and schedule(s) to be 
studied further in the expansion phase of the study, as well as in future phase 2 studies. 
The dose escalation portion of the study has evaluated dose regimens ranging from 2 to 7 
mg QD, 7 to 40 mg QW, 6 to 20 mg QDx3dQW, and 7 to 13 mg QDx5dQW 
(Investigator’s Brochure, 2017). 
 
Study INK128-002 
Study INK128-002 is evaluating safety and anti-tumor activity of MLN0128 (TAK-228) 
in subjects with hematologic malignancies (MM and WM). As of 09 December 2012, 39 
subjects have been treated in Study INK128-002. The most common AEs (≥ 20%), 
regardless of causality were nausea (56%), fatigue (49%), hyperglycemia (38%), 
thrombocytopenia (36%), diarrhea (28 %), decreased appetite and vomiting (23% each), 
and stomatitis/anemia (21% each). Most commonly reported (at least 2 subjects) Grade ≥ 
3 AEs, regardless of causality included thrombocytopenia (12%) and hypocalcemia, 
hypokalemia, and pneumonia (8 % each).  In Study INK128-002, dose escalation is 
completed, with 4 mg determined as the MTD for the QD schedule, and 9 mg determined 
as the MTD for the QDx3dQW schedule (Investigator’s Brochure, 2017). 
 
Study INK128-003 
Study INK128-003 is evaluating safety and anti-tumor activity of MLN0128 (TAK-228) 
in subjects with advanced solid tumors in combination with paclitaxel (and trastuzumab 
for HER2+ subjects). As of 09 December 2016, 67 subjects have been treated in Study 
INK128-003. The most common AEs (≥ 20%), regardless of causality were fatigue 
(67%); nausea (60%); diarrhea (52%); vomiting (46%), decreased appetite and 
hyperglycemia (43% each), stomatitis (40%), anemia and dehydration (37% each), 
asthenia (33%), hypokalemia (28%), neutropenia and urinary tract infection (27% each), 
decreased weight (22%), and dizziness and hypophosphatemia (21% each).  Most 
commonly reported (at least 2 subjects) Grade ≥ 3 AEs, regardless of causality, include 
neutropenia (21%); hypophosphatemia (15%); diarrhea and hyperglycemia (12% each), 
fatigue, hypokalemia, and vomiting (10% each) (Investigator’s  Brochure, 2017). 
 

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In Study INK128-003, dose escalation is completed, with 8 mg of MLN0128 (TAK-228) 
QDx3dQW being selected for the dose expansion phase of the study. The QDx5dQW 
and QW schedules were abandoned before MTDs were declared, as these schedules were 
viewed as less convenient relative to the QDx3dQW schedule, from the perspective of 
administering the paclitaxel and trastuzumab combination (Investigator’s Brochure, 
2017). 
 
Study MLN0128-1004 
Study MLN0128-1004 was a phase 1 open-label study to evaluate the safety, tolerability, 
and PK of TAK-228 as a single agent and in combination with paclitaxel in adult patients 
with advanced nonhematologic malignancies. As of the data cutoff date for the primary 
CSR (30 June 2016 ), 5 patients were ongoing in the study. The study had 3 arms that 
enrolled in parallel.  The MTD for QD dosing of milled TAK-228 was 3 mg TAK-228. The 
highest doses of milled TAK-228 that were tested and deemed safe in the QW and the 
QD×3d QW+paclitaxel cohorts of this study were 30 mg TAK-228 and 6 mg TAK-228, 
respectively.  
 
A total of 61 patients were enrolled in the study and received at least 1 dose of study drug: 
19 patients in the Single-Agent QD Arm, 22 patients in the Combination Arm, and 20 
patients in the Single-Agent QW Arm. 
 
Fifteen patients (78.9%) in the PK run-in period and all patients (100.0%) in the study 
treatment period of the Single-Agent QD Arm reported at least 1 TEAE. The most 
commonly reported (≥25%) preferred terms were vomiting (26.3%) during the PK run- in 
period and fatigue (58.8%); pruritus (47.1%); decreased appetite, diarrhea, and nausea 
(35.3% each); and insomnia (29.4%) during the study treatment period. Clear dose 
relationships were not evident among the most commonly reported TEAEs. 
 
Twenty-one patients (95.5%) in the Combination Arm reported at least 1 TEAE. The most 
commonly reported (≥25%) preferred terms were diarrhea (63.6%), nausea (54.4%), 
decreased appetite and fatigue (50.0% each), stomatitis and vomiting (36.4% each), and dry 
mouth (27.3%). Clear dose relationships were not evident among the most commonly 
reported TEAEs 
 
Nineteen patients (95.0%) in the Single-Agent QW Arm reported at least 1 TEAE. The 
most commonly reported (≥25%) preferred terms were fatigue and nausea (55.0% each), 
vomiting (45.0%), and diarrhea (30.0%). Clear dose relationships were not evident among 
the most commonly reported TEAEs (Investigator’s Brochure, 2017). 
 
Study C31001 
Study C31001 is an ongoing phase 1b/2 study of the safety and efficacy of TAK- 228 + 
exemestane or fulvestrant when administered in women with ER+/HER2- advanced or 
metastatic breast cancer that has progressed on prior treatment with everolimus in 
combination with exemestane or fulvestrant. Patients in this study receive the same prior 
regimen of either exemestane or fulvestrant that they received prior to enrolling in this 
study; the only change is that they also receive TAK-228 as part of their continued 

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regimen. 
 
As of the clinical data cutoff date (09 December 2016), 24 patients had received ≥1 TAK-
228 dose in the phase 1 portion of the study and 68 patients had received ≥1 TAK-228 
dose in the phase 2 portion of the study. Overall, most of the women enrolled as of the 
data cut were white (88% phase 1; 82% phase 2). At baseline, their median age was 
approximately58 years (range 32-83 years). As of data cutoff, 21 patients (2 in the phase 
1 portion and 19 in the phase 2 portion) remained ongoing in the study. 
 
As of the clinical data cutoff date, 10 treatment-emergent SAEs had been reported in 5 
patients (21%) in the phase 1 portion of Study C31001. One Grade 5 (fatal) SAE of 
hepatic failure was reported. One Grade 4 SAE (dyspnea) was reported and resulted in 
study drug discontinuation. 
 
As of the clinical cutoff date, 16 treatment-emergent SAEs had been reported in 11 
patients (16%) in the phase 2 portion of Study C31001. One Grade 5 (fatal) SAE was 
reported (preferred term: death [not further specified]). No Grade 4 SAEs were reported; 
the remaining events were severity Grade 3 or lower. 
 
Overall, across both phases of the study, commonly reported TEAEs included nausea 
(67% phase 1; 49% phase 2), fatigue (71% phase 1; 38% phase 2), diarrhea (67% phase 
1; 28% phase 2), hyperglycemia (25% phase 1; 26% phase 2), pruritis (50% phase 1; 
16% phase 2), vomiting (29% phase 1; 22% phase 2), and decreased appetite (33% phase 
1; 19% phase 2). TEAEs that were considered severity ≥Grade 3 and were reported in 
more than 3 patients were hyperglycemia (8 patients), diarrhea (7 patients), nausea (5 
patients), and fatigue (4 patients) (Investigator’s Brochure, 2017). 
 
Study C31002 
Study C31002 is a phase 1 open label, single-arm, multicenter study to evaluate the 
effect of a single dose of 40 mg TAK-228 on the QT/QTc (QT interval corrected for 
heart rate) in patients with advanced solid tumors. The data cutoff date for the primary 
study report was 29 April 2016. 
 
After completing the per-protocol PK/ECG assessments on Cycle 1 Day 3, patients 
could continue to receive TAK-228 if, in the opinion of the investigator, the patient was 
deriving clinical benefit, until they experienced disease progression. Patients continuing 
treatment received TAK-228 30 mg QW in 28-day cycles. 
 
The most frequently observed TEAEs by preferred term (>25% of patients) after 
treatment with TAK-228 were nausea (35 [80%] patients), fatigue (27 [61%] patients), 
vomiting (25 [57%] patients), decreased appetite (20 [45%] patients), diarrhea (15 
[34%] patients), weight decreased (14 [32%] patients), and urinary tract infection (12 
[27%] patients). 
 
 
 

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PK Summary 
PK data from Studies INK128-001, INK128-002, and INK128-003 indicate that TAK-
228 exhibits fast oral absorption (tmax generally between 1-4 hours after dosing) and 
dose-linear PK with a mean plasma half-life of approximately 8 hours and does not 
accumulate meaningfully in plasma when dosed as frequently as once daily and under 
any of 4 tested dosing regimens. The PK data of TAK-228 were generally consistent, 
with no appreciable differences across 5 phase 1 studies, suggesting no appreciable 
difference in the PK of TAK-228 among patients with advanced solid tumors or 
patients with MM or WM. 
 
There were no meaningful differences in the PK of TAK-228 when administered 24 
hours after a 30-minute IV infusion of 80 mg/m2 paclitaxel (Study INK128-003) 
compared with single-agent TAK-228 (Studies INK128-001 and INK128-002). The 
PK of paclitaxel also remained generally unaffected by TAK-228 co-administration, 
indicating the lack of a PK interaction between TAK-228 and paclitaxel. 
 
There were no readily apparent differences in either the Cmax or AUC of 4 mg TAK-228 
unmilled or milled capsules when administered under fasted conditions. 
 
Compared to the fasted state, when 4 mg of milled TAK-228 API was administered 
following a standard high-fat breakfast, there was an approximately 40% reduction in 
Cmax and a delay in tmax (median tmax 6 hours [fed] vs. 2 hours [fasted]), but there was no 
meaningful change in AUC. The differences observed when TAK-228 was dosed under 
fed versus fasted conditions may help explain the different MTDs determined for TAK-
228 QD dosing in Study INK128-001 compared with Study MLN0128-1004. 
 
There were no readily apparent differences in the PK of TAK-228 when administered 
in conjunction with either 25 mg exemestane or with 500 mg fulvestrant. 
 
 
Safety Issues 
As of the clinical data cutoff (09December 2016), a total of 538 patients had received 
≥ 1 dose of study drug across studies. A total of 25 deaths that occurred within 30 days of 
the last study drug dose had been reported to the clinical database as of the data cutoff; of 
these events, 1 (ventricular fibrillation and cardiac arrest; Study INK128-001) was 
considered related to TAK-228. 
Across the studies and regardless of causality or dosing regimen, the most common 
TEAEs included nausea, fatigue, hyperglycemia, vomiting, diarrhea, stomatitis, and 
decreased appetite. 
 
Due to the cardiac death on study INK128-001, study C31002, a phase 1 single-arm study 
to evaluate the effect of a single dose of 40 mg TAK-228 on the QT/QTc interval was 
initiated in patients with advanced solid tumors.  After completing the per-protocol 
PK/ECG/cardiac contractility monitoring, the patients continued MLN0128 (TAK-228) 
30 mg QW with continued cardiac monitoring.  The study results showed that treatment 
with MLN0128 (TAK-228) was not associated with clinically meaningful effects on the 
overall electrocardiographic safety profile, and that ECHO/MUGA at screening was not 

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required.   
 
Development of a Milled Formulation of MLN0128 (TAK-228) 
In order to allow more predictable absorption of MLN0128 (TAK-228) after oral 
administration and to allow scale-up manufacturing of MLN0128 (TAK-228) capsules, 
Millenium/Takeda developed a new milled formulation of the agent.  The physical 
milling step during the granulation process controls particle size distribution of 
MLN0128 (TAK-228).  In order to observe whether this milling step altered the safety 
and PK profile of MLN0128 (TAK-228), the company performed in vivo studies with PK 
analysis of milled agent.  These studies indicated that the milled formulation may result 
in faster absorption with possibly higher maximum concentration (Cmax), which could 
result in a different safety profile, compared to the previous unmilled API capsules. 
 
Takeda developed new MLN0128(TAK-228) capsules containing milled active 
pharmaceutical ingredient (API) for clinical studies in 1 mg, 3 mg, and 5 mg strengths.  
Patients receiving the milled formulation were added onto ongoing studies C31001 and 
C31002, as well as a new study MLN0128-1004, with various treatment cohorts 
including daily and weekly administration of milled MLN0128(TAK-228). 
 
The recommended dose of milled MLN0128 (TAK-228) was evaluated in 17 patients of 
MLN0128-1004, with PK, safety, and tolerability assessed.  Six patients were given a 4 
mg QD dose of milled MLN0128 (TAK-228) and 3 patients had observed DLT (rash, 
appetite loss and fatigue).  A dose of 3 mg QD was given to 11 patients with only 1 DLT 
(decreased platelets) observed.  The 3 mg QD dose of MLN0128 (TAK-228) was 
declared the RP2D, and was generally well tolerated and demonstrating objective 
responses in patients.   
 
The significant difference in tolerability observed in the comparison of the MTDs 
between unmilled and milled MLN0128 (TAK-228) when administered QD may be 
possibly explained due to the effect of food on the safety/tolerability of unmilled drug in 
study IND128-001.  The GastroPlusTM simulation performed under fasting conditions on 
the trial demonstrated that unmilled and milled MLN0128 (TAK-228) administration 
result in comparable exposures to the drug; whereas in the fed state, milled 
MLN0128(TAK-228) resulted in higher Cmax (1.5- to 2-fold higher) and earlier Tmax than 
unmilled MLN0128(TAK-228) with comparable AUCs.  Consequently, a dose of 3 mg 
QD was chosen as the RP2D of milled MLN0128 (TAK-228) dose in empty stomach 
conditions.   
 
The RP2D for milled MLN0128(TAK-228) on a weekly schedule was determined to be 
30 mg, the same weekly RP2D as seen for the older unmilled formulation.  Six patients 
treated at 30 mg weekly with the milled formulation did not demonstrate any DLT, but 
the agent was not escalated further.  No DLT had been demonstrated for milled 
MLN0128 (TAK-228) at the prior 20 mg QW dose as well.  
 
TAK-228-1004, a phase I, open label study to evaluate the safety, tolerability, and 
pharmacokinetics of MLN0128 (TAK-228) in combination with paclitaxel in adult 
patients with advanced non-hematological malignancies-), with the new milled API was 

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used to determine the recommended phase 2 dose (RP2D) for MLN0128(TAK-228) 
QD×3days per week in combination with paclitaxel.  The RP2D of milled MLN0128 
(TAK-228), given 3 consecutive days weekly, in combination with weekly paclitaxel at 
80 mg/m2, was 4 mg.   
 
2.3 
VEGF-Trap (Ziv-Aflibercept) 
 
Ziv-aflibercept is also referred to as “AVE0005,” “AVE0005 (VEGF Trap),” “VEGF Trap,” “V-
Trap,” and “VGFT” in various clinical study protocols and other documents. However, it will be 
referred to by its international non-proprietary name (INN), ziv-aflibercept, throughout this 
protocol (Investigator’s Brochure, 2017). 
 
Ziv-aflibercept is a recombinant fusion protein consisting of human vascular endothelial growth 
factor (VEGF) receptor extracellular domains fused to the Fc portion of human immunoglobulin 
G1 (IgG1). Ziv-aflibercept contains portions of the extracellular domains of 2 different vascular 
endothelial growth factor receptors (VEGFRs): VEGFR1 (also known as Flt-1) and VEGFR2 
(also known as KDR or Flk-1). Ziv-aflibercept binds VEGF in the picomolar (pmol/L) range, 
and also binds placental growth factor (PlGF), although with lower affinity. The affinity 
constants (KD) for binding to 2 human isoforms of VEGF, VEGF165 and VEGF121, are 0.50 
pmol/L and 0.36 pmol/L, respectively. The KD for human PlGF2 is 39 pmol/L. The binding of 
ziv-aflibercept to its ligands in vivo is expected to block tumor angiogenesis and vascular 
permeability (Investigator’s Brochure, 2017). 
 
Ziv-aflibercept drug product is formulated as a sterile liquid for intravenous (IV) administration. 
Ziv-aflibercept has been found to be active with a broad pharmacological index against early and 
advanced stage disease in a variety of preclinical solid tumor models including sarcomas, and 
ovarian, prostate, mammary, colon, and gastric carcinomas when used as a single agent or in 
combination with cytotoxic agents. In mouse models of ascites formation with ovarian and renal 
cell carcinoma, ziv-aflibercept inhibited ascites formation and reduced tumor burden 
(Investigator’s Brochure, 2017). 
 
Two analytes were assayed in animal models specifically by enzyme linked immunosorbent 
assay (ELISA) methods: free aflibercept (compound not complexed to VEGF), and bound 
aflibercept (complexed aflibercept: VEGF [ratio 1/1]) (Investigator’s Brochure, 2017). 
 
Following IV administration in all animal species evaluated, free aflibercept was characterized 
by a low clearance (0.5 to 3 mL/hr/kg), a low volume of distribution (51 to 77 mL/kg), and a 
long apparent elimination half-life (t1/2) of 48 to 98 hours. Based on the correlation between 
exposure and activity in non-clinical models, the target pharmacological exposure in humans is 
proposed to be a safely administered dose of ziv-aflibercept at which an excess of free 
aflibercept is sustained (Investigator’s Brochure, 2017). 
 
Mechanism of Action 
 
VEGF is an angiogenic growth factor that plays a critical role during normal embryonic 
development and has also been implicated in the pathogenesis of a number of diseases, 
including cancer.  The angiogenic growth factor family, comprised of 5 isoforms (VEGF- A, -B, 
-C, -D, -E, and placental growth factor [PlGF]), acts through 3 tyrosine kinase receptors, 

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VEGFR1, VEGFR2 and VEGF receptor 3 (VEGFR3). The diagram of VEGF Trap (below) 
shows the high affinity VEGF binding domain 2 from VEGFR1 coupled with the biologically 
more active VEGFR 2 domain 3 along with the IgG1 Fc domain. With a dissociation constant of 
0.5 pM, the VEGF Trap has the highest binding affinity for VEGF described to date. 
 
VEGF Trap is produced in Chinese hamster ovary cells as a dimeric 
glycoprotein with a molecular weight of 115 kDa (Holash et al.,  2002).  
Unlike the humanized monoclonal antibody to VEGF, VEGF Trap is 
entirely comprised of human protein sequences, has a higher affinity for 
binding to VEGF, and binds to other VEGF family members, including 
PlGF.  In addition, VEGF Trap has been shown to block VEGFR2 
phosphorylation when added to cultured endothelial cells in the presence of 
exogenous VEGF (Holash et al.,  2002). 
 
 
Nonclinical Studies with Ziv-Aflibercept in Human Tumor Models 
 
The toxicological profile of ziv-aflibercept was established via single and repeat dose toxicity 
studies in rats (general toxicity and safety pharmacology), monkeys (general toxicity and 
fertility) and rabbits (reproductive toxicity and local tolerance) (Investigator’s Brochure, 2017). 
 
Data from the general toxicity studies conducted in cynomolgus monkeys showed that most of 
the adverse effects of ziv-aflibercept were consistent with the inhibition of the VEGF pathway 
and were also reported in nonclinical studies conducted with other anti- VEGF drugs. Target 
organs identified in these studies included bone (interference with growth plate maturation of 
long bones and osteocartilaginous exostoses of vertebrae), kidney (frequently increased 
glomerular mesangial matrix, occasionally hyperplasia of parietal epithelium and periglomerular 
fibrosis), adrenals (decreased vacuolation with eosinophilia in the zona fasciculata), ovary 
(decreased number of maturing follicles, granulosa cells and/or theca cells), and nasal cavity 
(atrophy/loss of the septum and/or turbinates associated with necrotizing inflammation and 
various other epithelial, microvascular, cartilaginous and osseous findings). Other microscopic 
findings in the 6- month chronic toxicity study included vascular alterations in the choroid 
plexus and digestive tract, vascular degeneration and fibrosis in several tissues including the 
heart, and hepatic portal inflammation and periportal necrosis (Investigator’s Brochure, 2017). 
 
Ziv-aflibercept was shown to alter fertility parameters in sexually mature male and female 
monkeys treated for 6 months. These effects were noted at plasma exposures of free aflibercept 
that were close to exposures in patients at the recommended pharmacological doses and were 
considered to impact fertility in both males and females. Inhibition of the female reproductive 
function by anti-VEGF drugs is related to the key role of VEGF-mediated angiogenesis in 
follicular and luteal development. The effects of ziv-aflibercept on sexually mature male and 
female fertility were reversible (Investigator’s Brochure, 2017). 
 
The developmental effects of ziv-aflibercept were evaluated in an embryo-fetal study in rabbits. 
Ziv-aflibercept induced embryo-toxicity at maternotoxic doses and teratogenic effects at all 
doses tested. Ziv-aflibercept was locally well tolerated in rabbits receiving a single injection by 
the IV, intramuscular, and subcutaneous (SC) routes (Investigator’s  Brochure, 2017). 

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Ziv-Aflibercept Nonclinical Toxicology and PK studies 
 
The safety pharmacology studies showed that ziv-aflibercept reduced vascular density in certain 
normal tissues in mice, increased blood pressure in rats and mice and delayed wound repair and 
healing in rabbits. Detectable effects on some of these parameters were noted at doses of ziv-
aflibercept that were lower than the optimal pharmacological doses determined in tumor bearing 
mice. Maximal effects on wound healing and blood pressure were noted only at doses in the 
same range as pharmacological active doses. The increase in blood pressure reported in rodents 
was reversible and was reported in patients treated with ziv-aflibercept. The reversibility of the 
effect on wound repair and healing was not evaluated. These effects are related to the inhibition 
of the VEGF pathway. Ziv- aflibercept had no effects on respiratory function in rats or on 
thrombus formation in rabbits. Based on results obtained from toxicology studies, the absence 
of major effects on the central nervous system and cardiac function was established 
(Investigator’s  Brochure, 2017). 
 
Summary of Ziv-Aflibercept Clinical Studies 
 
As of 03 August 2016, the clinical development of ziv-aflibercept encompasses a total of 51 
clinical oncology trials (31 Sanofi trials and 20 National Cancer Institute [NCI] trials) in more 
than 4385 cancer patients mostly with advanced solid malignancies as well as 2 clinical trials in 
76 healthy subjects (PDY6655 and PDY6656).  Doses have been administered up to 800 μg/kg 
SC twice weekly, 7 mg/kg IV every 2 weeks (q2w), and 9 mg/kg IV every 3 weeks (q3w) 
(Investigator’s Brochure, 2017). 
 
Ziv-aflibercept was initially administered SC in 2 single-agent dose-escalation safety and 
pharmacokinetic (PK) studies (TED6113/6114) in patients with advanced solid tumors or 
lymphomas. However, the SC route of administration was discontinued due to the high volume 
of injection required for the formulated ziv-aflibercept concentration of 25 mg/mL. 
Subsequently, only IV administration was used in all clinical studies (Investigator’s Brochure, 
2017). 
 
Phase 1 single-agent dose escalation studies (TED6115/TED6116) evaluated safety, tolerability, 
PK and anti-tumor activity in patients with various solid tumor types. Phase 2 single-agent 
studies were designed to measure the effect of ziv-aflibercept in patients with ovarian cancers 
(ARD6122, ARD6772, and EFC6125), and non-small-cell lung adenocarcinoma ([NSCLA], 
ARD6123) (Investigator’s Brochure, 2017). 
 
Combination Phase 1 dose-escalation studies were conducted to evaluate safety, PK, and 
antitumor activity in patients with solid malignancies. The recommended Phase 2 dose (RP2D) 
of ziv-aflibercept was confirmed in these Phase 1 combination studies as 4 mg/kg every 2 weeks 
(q2w) when combined with chemotherapy administered every 2 weeks, as was initially 
determined in single-agent studies TED6115/TED6116. 
 
Combination Phase 3 studies’ primary objective was efficacy. In these studies, ziv-aflibercept 
was administered IV over 1 hour at 4 mg/kg q2w or 6 mg/kg q3w depending on the associated 
chemotherapy schedule (Investigator’s Brochure, 2017). 
 

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In the q2w regimen in Phase 1 and Phase 3 combination studies, ziv-aflibercept was combined 
with standard doses of the following cytotoxic agents: oxaliplatin/5- fluorouracil/leucovorin 
(FOLFOX4), [TCD6117]), irinotecan/leucovorin/5-fluorouracil (irinotecan/LV5FU2, 
[TCD6118]), and gemcitabine or gemcitabine and erlotinib (TCD6121), gemcitabine 
(EFC10547/VANILLA), and irinotecan/5-FU/leucovorin (FOLFIRI, [EFC10262/VELOUR]). 
In the q3w regimen in Phase 1 and Phase 3 combination studies, ziv-aflibercept was combined 
with standard doses of docetaxel (Taxotere®)/cisplatin/5-fluorouracil (TCF, [TCD6119]), 
docetaxel or docetaxel/cisplatin or pemetrexed (TCD6120), and docetaxel (EFC10261/VITAL) 
(Investigator’s Brochure,  2017). 
 
Five Phase 3 combination studies have been conducted in the following indications: pancreatic 
(EFC10547/VANILLA), lung (EFC10261/VITAL) metastatic colorectal cancer ([MCRC], 
EFC10262/VELOUR and EFC11338/AFLAME), and prostate (EFC6546/VENICE). At the IB 
cutoff, all were completed (Investigator’s Brochure, 2017). 
 
In addition, 19 studies are being conducted with ziv-aflibercept under the sponsorship of the 
U.S. NCI cooperative group. Lastly, other studies have been conducted with a different 
formulation of aflibercept (VEGF-Trap Eye) in the ophthalmology indication (41 completed 
studies), under separate sponsorship. A biologics license application was authorized to 
Regeneron Pharmaceuticals for tradename EYLEA for treatment of patients with neovascular 
(wet) agerelated macular degeneration by the US FDA in November 2011(Investigator’s 
Brochure, 2017). 
 
Study EFC10262/VELOUR was a multinational, randomized, double-blind study, comparing 
the efficacy of ziv-aflibercept q2w versus placebo in patients with MCRC treated with 
irinotecan /5-FU combination (FOLFIRI) after failure of an oxaliplatin based regimen. The 
safety population consisted of 1216 MCRC patients who received either ziv-aflibercept plus 
FOLFIRI (611 patients) or placebo plus FOLFIRI (605 patients) (Investigator’s Brochure, 
2017). 
 
The efficacy conclusion from EFC10262/VELOUR was that 4 mg/kg ziv-aflibercept 
administered as a 1-hour IV infusion q2w in combination with the FOLFIRI regimen 
demonstrated a statistically and clinically significant improvement in overall survival (OS), 
progression free survival (PFS) duration and a significantly higher overall response rate (ORR) 
in patients treated with ziv-aflibercept/FOLFIRI over those treated with placebo/FOLFIRI. 
These results were consistent across prespecified subgroups (gender, ethnic origin, age, body 
weight, creatinine clearance, Eastern cooperative oncology group [ECOG] performance status 
[PS] at baseline) and their robustness was confirmed by sensitivity analyses (Investigator’s 
Brochure, 2017). 
 
Overall, considering safety results from EFC10262/VELOUR, the addition of ziv-aflibercept to 
FOLFIRI gave no new toxicity signals but showed that: Some AEs of FOLFIRI were enhanced: 
These included grade 1 or 2 events such as diarrhea, asthenic conditions, decrease appetite, 
weight decrease, palmar plantar erythrodysesthesia syndrome, and hematological abnormalities 
(with the exception of anemia) and grade 3 or 4 events (>2% higher incidence in ziv-
aflibercept/FOLFIRI arm compared to placebo/FOLFIRI arm) such as neutropenia, 

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hypertension, diarrhea, asthenic conditions, leukopenia, stomatitis and ulceration, infection, 
neutropenic complications and gastrointestinal (GI) and abdominal pains (Investigator’s 
Brochure, 2017). 
 
The risk of treatment-emergent adverse events (TEAEs) identified as possible anti-VEGF class 
specific AE (analyzed by grouped terms) were significantly increased and these were more 
frequently all grade hypertension and hemorrhage TEAEs. Considering analysis per prespecified 
subgroups during EFC10262/VELOUR, no unexpected clinical important difference was seen 
across the various analyzed subgroups (gender, race, age, of body weight, creatinine clearance, 
ECOG PS at baseline) with regard to incidences of TEAEs in patients treated with ziv-
aflibercept/FOLFIRI. Similarly for the grouped terms of particular interest (hypertension, 
hemorrhage, venous thromboembolic events [VTEs], arterial thromboembolic events [ATEs], 
wound healing complications, renal failure events, acute drug reactions) regression analyses 
showed that interaction terms of treatment and the various intrinsic factors were not statistically 
significant indicating that the incidence of the selected grouped terms were not significantly 
different across the studied subgroups. The safety profile (either VEGF-blockade AEs or 
chemotherapy induced toxicities) in patients with renal or liver function impairment did not 
significantly differ when compared to that observed in patients with normal renal or liver 
function at baseline. Lastly, the safety profile of ziv-aflibercept combined with FOLFIRI was 
comparable between patients with or without prior bevacizumab (Investigator’s  Brochure, 
2017). 
 
Safety Issues 
 
Overall, consistency was found between safety findings observed in the pivotal 
EFC10262/VELOUR trial and supporting safety data obtained from pooled analysis of single 
agent Phase 1 and Phase 2 studies (TED6115/6116, ARD6122, ARD6123, ARD6772, 
EFC6125), Combination Phase 1 studies analyzed side by side (TCD6117, TCD6118, 
TCD6119, TCD6120, TCD6121), and other Phase 3 studies analyzed individually 
(EFC10547/VANILLA, EFC10261/VITAL, and EFC6546/VENICE) (Investigator’s Brochure,  
2017). 
 
In the pooled analysis of single-agent Phase 1 and Phase 2 studies for ziv-aflibercept, there was 
no apparent dose dependent relationship between 2 and the 4 mg/kg with regard to safety 
profile. The most frequently reported TEAEs by PT in both dose level groups were asthenia and 
fatigue, hypertension, headache, gastrointestinal pains (abdominal pain, abdominal pain upper), 
nausea, dysphonia, vomiting, arthralgia, decreased appetite, constipation, breathing 
abnormalities (cough, dyspnea), diarrhea, and arthralgia. All events occurred with an incidence 
of ≥20% overall, in the 2 and 4 mg/kg groups, except for dyspnea and cough in the 2 mg/kg 
group (17.0% and 11.6%, respectively), and for arthralgia in the 4 mg/kg group (18.2%) 
(Investigator’s Brochure, 2017). 
 
In combination studies for q2w and q3w regimens, in addition to the most commonly reported 
TEAEs in single-agent studies, the comparison of the rate of TEAEs (all grade) across Phase 1 
combination studies (both regimens) showed the same categories of TEAEs (PT or HLT term 
≥20%) than those observed in single-agent studies but with higher percentages when focusing 
on the expected background chemo-induced toxicities (such as diarrhea, nausea, vomiting, 

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decreased appetite, asthenic conditions [fatigue and anemia]). Infections percentage was also 
increased except in the gemcitabine and pemetrexed combinations. Other TEAEs were observed 
with an incidence ≥20% in Phase 1 combination studies in both regimen groups: Stomatitis and 
ulceration (HLT; except in the gemcitabine, pemetrexed and FOLFOX combinations), alopecia 
(only for combination with irinotecan/LV5FU in q2w regimen), pyrexia, palmar plantar 
erythrodysesthesia syndrome and weight decreased. In addition, some differences were 
observed between the 2 regimen groups depending on the background chemotherapy associated 
to ziv-aflibercept, such as dysgeusia, palmar-plantar erythrodysaesthesia, nail disorder in the 
q3w regimen studies (mainly in docetaxel-based combinations) specifically reported with an 
incidence ≥20% (Investigator’s Brochure, 2017). 
 
2.4 
Rationale for Combination of MLN0128 (TAK-228) and VEGF-Trap (Ziv-
Aflibercept) 
 
Angiogenesis is one of the cardinal processes leading to invasion and metastasis of solid tumors.  
Synergistic effects of mTOR inhibitors combined with antiangiogenic agents have been widely 
researched and supported by extensive clinical and preclinical data. The inhibition of the mTOR 
pathway has been proven to exhibit antiangiogenic effects, further strengthening the capabilities 
of angiogenesis inhibitors such as ziv-aflibercept (Hanahan and Weinberg, 2011). 
 
The angiogenic-signaling pathway may be triggered by the release of angiogenic promoters such 
as VEGF from tumor cells into the local microenvironment (Alvarez et  al., 2012).  VEGF is 
largely upregulated by the presence of hypoxia-inducible factor 1α (HIF-1α).  HIF-1α mediates 
adaptive responses to hypoxic conditions.  Increase in HIF- 1α in response to a hypoxic 
environment is critical to the establishment and progression of many common cancers.  
Consequently, HIF-1-dependent activation of genes allows cancer cells to survive and 
metastasize. 
 
Increased HIF-1α is associated with increased expression of VEGF, aggressive tumor growth, 
and poor patient prognosis.  HIF-1α inhibition in combination with antiangiogenic therapy is a 
promising strategy for targeting tumor resistance (Naing,  2013). Agents that inhibit the mTOR 
pathway, like MLN0128 (TAK-228), have been shown to inhibit the activity of several 
angiogenic factors, including HIF-1α, which in turn results in decreased VEGF, thus decreased 
angiogenic activity (Majumder, et al., 2004). In a phase III study of everolimus in pancreatic 
neuroendocrine tumors, Yao et al. has demonstrated that mTOR inhibition may reduce 
circulating levels of sVEGFR1, PlGF, and bFGF (Yao et al., 2011).  This important property of 
mTOR inhibitors, such as MLN0128 (TAK-228), makes them ideal candidates for 
combination therapy with ziv-aflibercept. 
 
Previous studies of combination bevacizumab and temsirolimus resulted in higher overall 
response rate; however, there were absolutely no differences in progression free survival and the 
toxicity profile of the combination of temsirolimus and bevacizumab. The full doses of each 
drug were much higher than anticipated and limited treatment continuation over time (Heng, 
2011). 
 
Our proposal of combining MLN0128 (TAK-228) and ziv-aflibercept have broader target 

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inhibition than bevacizumab/rapalog (TORC21, PlGF, etc.), and therefore the risk of toxicities 
could be higher. The advantage of our trial is it could prospectively define a few parameters for 
the measurement of tolerability, not just MTD. We will consider parameters such as rate of 
dose delay or dose reduction throughout treatment; rate of treatment discontinuation due to dose 
delay or dose reduction throughout treatment; and percentage of patients that can tolerate 
therapy through 2 months, 4 months, 6 months, etc. 
 
Safety Issues 
 
As of December 31, 2014, there had been five patients treated on this protocol. The first dose 
level explored was MLN0128 (TAK-228) 3 milligrams PO daily and ziv-aflibercept 3 
milligram/kilogram intravenously every 2 weeks starting on Cycle 1 Day 1.  The Grade1-2 
possibly-related toxicities noted in the first 5 patients were as follows: arthralgia, mucositis, rash, 
hyperglycemia, hypercholesterolemia, hypertriglyceridemia, hypertension, abdominal pain, 
anorexia, weight loss, proteinuria, thrombocytopenia, elevated alanine aminotransferase, 
elevated aspartate aminotransferase, nausea, vomiting, dehydration, and diarrhea. The Grade 3 
possibly-related events noted in the first 5 patients were pancreatitis (n=2) and rash (n=2). There 
have been 2 dose-limiting toxicities in the first dose level: rash maculo-papular and pancreatitis. 
 
Based on internal data and discussion between CTEP and Millennium, MLN0128 (TAK-228) is 
better tolerated when given intermittently such as 3 days on and 4 days off, and the highest 
tolerated dose with that schedule has been 9 mg. Since we have seen dose-limiting toxicity on 
the first dose level, a more conservative approach has been implemented in the Amendment 06 
dosing schema, in which patients will begin MLN0128 (TAK-228) on Day 2 of Cycle 1 and 
receive it for 3 days on and 4 days off.  Ziv-aflibercept will continue to be given on Days 1 and 
15 of each cycle. 
 
 
2.5 
Correlative Studies Background 
 
Despite progress, the biologic activity of ziv-aflibercept may be difficult to assess because it 
may often seem primarily cytostatic rather than cytotoxic in function. When anti-VEGF agents 
are used in a monotherapeutic fashion, only a small number of patients have a major tumor 
response (Beaudry et al., 2005; Davis et al., 2003). To advance the clinical testing of ziv-
aflibercept and the projected additional benefits of combination with MLN0128 (TAK-228), we 
plan to conduct biopsies. These studies are designed to determine if combining ziv-aflibercept 
and MLN0128 (TAK-228) will inhibit both the phosphorylation of effectors that are 
downstream of the targets of the drugs as well as potentiating anti- angiogenic activities. 
 
For the expansion cohorts, we will be performing correlative studies. Our interventional 
radiologists will coordinate with our study radiologist. Our study radiologist will identify an 
optimal area for interventional radiologists to biopsy.  Biopsies will be performed at baseline, 
on Cycle 1 Day 8, and at time of progression. All the treating investigators will be educated and 
trained to report to the principal investigator the first signs of progression. 
 
Reverse Phase Protein Arrays (RPPA) 
 

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In collaboration with Dr. Funda Meric-Bernstam (Chair of the Department of Investigational 
Cancer Therapeutics; Professor, Department of Surgical Oncology, University of Texas MD 
Anderson Cancer Center), RPPAs will be performed on biopsy specimens, peripheral blood 
mononuclear cells (PBMC), and platelet-enriched plasma samples to quantitate protein 
expression, including phosphorylation, as well as markers of proliferation, cell cycle 
progression, angiogenesis and apoptosis.  Expression/ phosphorylation of potential markers of 
response, including phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-mTOR (Ser2448), 
total S6K1, phospho-S6K1 (Thr389), phospho-S6 (ser235/236), phospho-4E-BP1 (Thr70), 
phospho-4E-BP1 (ser65),; CD31, IGF-1R, insulin receptor will be assessed by RPPA. Array 
technology, robotics and robust algorithms are available to efficiently utilize the technologies as 
demonstrated in published work by Dr. Gordon Mills, Dr. Funda Meric-Bernstam, and their 
colleagues who have established and validated the technology at MD Anderson. 
 
Immunohistochemistry (IHC) Analysis 
 
In collaboration with Dr. Funda Meric-Bernstam, we will evaluate protein expression (both total 
and phosphorylated proteins) by IHC staining on slides from tumor samples. Expression of 
PI3K/mTOR signaling markers phospho-Akt (Ser473), phospho-S6 (ser235/236), phospho-4E-
BP1 (ser65), as well as PTEN, SCD1, IGF-1R, and insulin receptor will be assessed (Figure 3). 
Microvessel density will also be evaluated in the tumor tissue using IHC and correlated with 
clinical tumor response and RPPA analyses. Pre- and post-treatment biopsies will be evaluated 
by H&E.  Treatment-induced apoptosis will be assessed with IHC for cleaved caspase 3 and 
proliferation will be assessed by Ki- 67. 
 
Figure 3. Examples of positive and negative staining PI3K/mTOR markers by IHC. 
IHC for PTEN, pAkt Ser473, p4E-BP1 Thr70, pS6 Ser235/236, pS6 Ser240/244 and Ki- 
67.  Breast cancer samples with positive immunostaining are shown in the top panel, and 
samples with negative or low immunostaining are shown in the bottom panel. 
Data 
provided by Dr. Funda Meric-Bernstam. 
 
In the event that tissue material is limited, we will give priority to RPPA analysis as RPPA is an 
extremely effective and sensitive method for measuring multiple proteins in cancer cells.  This 
technique can be applied to minute amounts of material.  Dr. Funda Meric-Bernstam and Dr. 
Gordon Mills have established this method, and we are confident that we can use this 
technology to measure all the markers of relevance to this study.  However, when it is possible 

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to perform both RPPA and IHC for the same proteins, the results from one will validate the 
results from the other. 
 
 
 
3. PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
3.1.1 Patients with advanced or metastatic cancer that is refractory to standard therapy or 
relapsed after standard therapy. Patients must have histologically confirmed 
malignancy that is metastatic or unresectable and for which standard curative or 
palliative measures do not exist or are no longer effective. 
 
3.1.2 Patients enrolled in the expansion cohort must have biopsiable disease. There will 
be preferential enrollment of patients with pancreatic neuroendocrine tumors, 
fibrolamellar hepatocellular carcinoma, or ovarian 
cancer during the dose 
expansion cohort. 
 
3.1.3 Patients must be >4 weeks beyond treatment of any chemotherapy, other investigational 
therapy, hormonal, biological, targeted agents or radiotherapy, and must have recovered 
to < grade 1 toxicity or previous baseline for each toxicity. Exception: Patients may 
have received palliative low dose radiotherapy to the limbs 1-4 weeks before this 
therapy provided pelvis, sternum, scapulae, vertebrae, or skull were not included in the 
radiotherapy field. 
 
3.1.4 Age ≥18 years.  Because no dosing or adverse event data are currently available on 
the use of MLN0128 (TAK-228) in combination with ziv-aflibercept in patients <18 
years of age, children are excluded from this study, but will be eligible for future 
pediatric trials. 
 
3.1.5 ECOG performance status ≤1, see Appendix A. 
 
3.1.6 Life expectancy of greater than 3 months. 
 
3.1.7 Patients must have normal organ and marrow function as defined below: 
 
− 
leukocytes 
≥3,000/mcL 
− 
absolute neutrophil count 
≥1,500/mcL 
− 
platelets 
≥100,000/mcL 
− 
hemoglobin 
≥ 9 g/dL 
− 
total bilirubin 
≤1.5 × institutional upper limit of 
normal AST(SGOT)/ALT(SGPT) 
≤2.5 × institutional upper 
limit of normal 
− 
creatinine 
≤1.5 × institutional upper limit of 
normal OR 

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32 
 
− 
creatinine clearance 
≥60 mL/min for patients with creatinine levels 
above institutional normal. 
− 
Fasting serum glucose 
≤ 130 mg/dL 
− 
Fasting triglycerides 
≤ 300 mg/dL 
− 
HBA1C 
< 7.0% 
 
3.1.8 Patients must have evaluable or measureable disease by RECIST 1.1. 
 
3.1.9 Women of child-bearing potential MUST have a negative serum or urine pregnancy test 
within 7 days unless prior hysterectomy or menopause (defined as 12 consecutive 
months without menstrual activity). Patients should not become pregnant or breastfeed 
while on this study.  The effects of MLN0128 (TAK-228) and ziv-aflibercept on the 
developing human fetus are unknown.  For this reason, women of child-bearing 
potential must agree to use 1 highly effective method of contraception and 1 additional 
effective (barrier) method, at the same time, from the time of signing the informed 
consent through 90 days (or longer, as mandated by local labeling [eg; USPI, SmPC, 
etc;]) after the last dose of study drug; or agree to practice true abstinence.  
Should a woman become pregnant or suspect she is pregnant while she or her partner 
is participating in this study, she should inform her treating physician immediately.  
Male patients, even if surgically sterilized (i.e., status post-vasectomy), who: 
• Agree to practice highly effective barrier contraception during the entire study 
treatment period and through 120 days after the last dose of study drug, or 
• Agree to completely abstain from heterosexual intercourse. 
 
3.1.10 Ability to understand and the willingness to sign a written informed consent document. 
 
3.1.11 Ability to swallow oral medications 
 
 
 
3.2 
Exclusion Criteria 
 
3.2.1 Patients who have had chemotherapy or radiotherapy within 4 weeks (6 weeks for 
nitrosoureas or mitomycin C) prior to entering the study or those who have not 
recovered to </= grade 1 adverse events due to agents administered more than 4 weeks 
earlier. 
 
3.2.2 Patients who are receiving any other investigational agents. 
 
3.2.3 Patients with known brain metastases should be excluded from this clinical trial 
because of their poor prognosis and because they often develop progressive 
neurologic dysfunction that would confound the evaluation of neurologic and other 
adverse events. 
 
3.2.4 History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to MLN0128 (TAK-228) or ziv-aflibercept. 
 

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3.2.5 Uncontrolled intercurrent illness including active infection. 
 
3.2.6 Pregnant women are excluded from this study because MLN0128 (TAK-228) and ziv-
aflibercept are agents with the potential for teratogenic or abortifacient effects. 
Because there is an unknown but potential risk for adverse events in nursing infants 
secondary to treatment of the mother with MLN0128 (TAK-228) and ziv-aflibercept, 
breastfeeding should be discontinued if the mother is treated with MLN0128 (TAK-
228) and ziv-aflibercept.  These potential risks may also apply to other agents used in 
this study. 
 
3.2.7 Patients with known human immunodeficiency virus infection are not to be enrolled 
in the study. 
 
3.2.8 History of abdominal fistula, gastrointestinal perforation or intra-abdominal 
abscess 
within 28 days or manifestations of malabsorption due to prior 
gastrointestinal (GI) surgery or GI disease that may alter the absorption of 
MLN0128 (TAK-228). 
 
3.2.9 New York Heart Association Class III or greater congestive heart failure within last 6 
months or uncontrolled hyperlipidemia (cholesterol > 300 mg/dl; triglyceride 
2.5XULN despite lipid lowering agent) within last 3 months, see Appendix B. 
 
3.2.10 Uncontrolled diabetes (fasting serum glucose>130 mg/dl) despite best medical 
management or poorly controlled diabetes mellitus defined as HbA1c > 7%; subjects 
with a history of transient glucose intolerance due to corticosteroid administration are 
allowed in this study if all other inclusion/exclusion criteria are met. 
 
3.2.11 History of uncontrolled hypertension, defined as blood pressure > 150/95 mmHg, 
or systolic blood pressure >180 mmHg when diastolic blood pressure < 90 mmHg, 
on at least 2 repeated determinations on separate days within 3 months prior to 
study enrollment. 
 
3.2.12 Urine protein should be screened by dipstick or urine analysis. For proteinuria > 1+ 
or urine protein: creatinine ratio > 1.0, 24-hour urine protein should be obtained 
and the level should be < 2000 mg for patient enrollment. 
 
 
3.2.13 Patients on anticoagulant therapy with unstable dose of warfarin and/or having an out-
of- therapeutic range INR (>3) within the 4 weeks prior to drug administration. 
 
3.2.14 Evidence of clinically significant bleeding diathesis or underlying coagulopathy, 
non- healing wound. 
 
3.2.15 History of any of the following within the last 6 months prior to study entry: 
o Ischemic myocardial event, including angina requiring therapy and 
artery revascularization procedures 

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o Ischemic cerebrovascular event, including TIA and artery 
revascularization procedures 
o Requirement for inotropic support (excluding digoxin) or serious (uncontrolled) 
cardiac arrhythmia (including atrial flutter/fibrillation, ventricular fibrillation or 
ventricular tachycardia) 
o Placement of a pacemaker for control of rhythm 
o Pulmonary embolism 
3.2.16 Significant active cardiovascular or pulmonary disease at the time of study 
entry, including: 
o Uncontrolled high blood pressure (i.e., systolic blood pressure >150 mm 
Hg, diastolic blood pressure > 95 mm Hg) 
o Pulmonary hypertension 
o Uncontrolled asthma or O2 saturation < 90% by pulse oximetry on room air 
o Significant valvular disease; severe regurgitation or stenosis by imaging 
independent of symptom control with medical intervention, or history of 
valve replacement 
o Medically significant (symptomatic) bradycardia 
o History of arrhythmia requiring an implantable cardiac defibrillator 
 
3.2.17 Baseline prolongation of the rate-corrected QT interval (QTc) (e.g. repeated 
demonstration of QTc interval >480 milliseconds, or history of congenital long 
QT syndrome, or torsades de pointes) 
 
3.2.18 Psychiatric illness/social situations that would limit compliance with study requirements 
 
3.2.19 Have initiated treatment with bisphosphonates less than 30 days prior to the first 
administration of MLN0128 (TAK-228). Concurrent bisphosphonate use is only 
allowed if the bisphosphonate was initiated at least 30 days prior to the first 
administration of MLN0128 (TAK-228). 
 
3.2.20 Patients who are taking proton pump inhibitor (PPI) within 7 days before 
receiving the first dose of study drug or who require treatment with PPIs 
throughout the trial or those who are taking H2 receptor antagonists within 24 
hours of the first dose of study drug. 
 
3.2.21 Patients with known history of hepatitis B surface antigen-positive, or known 
history or suspected active hepatitis C infection are not to be enrolled in the 
study. 
 
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial. 

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4. REGISTRATION PROCEDURES 
 
Not applicable to this 
study. 
 
4.1 
General Guidelines 
 
Not applicable to this 
study. 
 
4.2 
Registration Process 
 
Not applicable to this 
study. 
 
 
5. TREATMENT PLAN 
 
This is a phase I, single center, open-label, dose-escalation study of MLN0128 (TAK-228) and 
ziv-aflibercept administered in combination to patients with metastatic or locally advanced 
solid tumors.  Additional sites may be added upon activation of NCI Early Experimental 
Therapeutics Network in consultation with CTEP. 
 
This study will follow a standard 3+3 design. Three patients will be treated per dose level. If 
none of the patients experience a dose-limiting toxicity (DLT), the next cohort of three 
patients will be treated at the next higher dose level. If 1 of 3 patients treated at a dose 
experiences a DLT, that cohort will then be expanded to a total of six patients. If a DLT 
occurs in only 1 in 6 of the first dose level, 3 more patients may be enrolled at the next 
higher dose level (i.e., cohort 2). If 2 or more of 6 patients in any cohort experiences DLT, 
the MTD is considered to have been exceeded. No patients may be enrolled in the next dose 
level until 3 patients at the previous dose level have completed at least one cycle of therapy. 
Subjects enrolled in the dose escalation cohort who do not receive 75% of each drug in the 
first cycle of treatment, for reasons other than DLT, will be considered not evaluable for 
DLT and will be replaced. Those patients that are not likely to comply with the protocol in 
the future may be replaced. 
 
Once the MTD is determined, or at the maximum tolerated dose level explored (Level 3) (if 
MTD is not reached at dose level 3), an additional 10 patients may enroll in the dose 
expansion cohort. Patients enrolled in this cohort must have biopsiable disease. We will be 
emphasizing enrollment of pancreatic neuroendocrine tumor (PNET), fibrolamellar 
hepatocellular carcinoma, and ovarian cancer patients. 
 
5.1 
Agent Administration 
 
All protocol-specific criteria for administration of MLN0128 (TAK-228) must be met and 
documented before drug administration.  The study drug will be administered only to eligible 

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patients under the supervision of the investigator or identified subinvestigator(s). 
 
Treatment will be administered on an outpatient basis.  Reported adverse events and potential 
risks are described in Section 7.  Appropriate dose modifications are described in Section 6. 
No investigational or commercial agents or therapies other than those described below may 
be administered with the intent to treat the patient's malignancy. 
 
One cycle consists of 4 weeks of treatment (28 days). MLN0128 (TAK-228) will be taken 
orally for 3 days on and 4 days off starting on Cycle 1 Day 2 (and starting Day 2 of every 
cycle), and ziv-aflibercept will be given via intravenously (IV) infusion once every 2 weeks 
(days 1 and 15 for each cycle). Both study drugs will have a +/- 2 day window. If the ziv-
aflibercept dosing is rescheduled, then the MLN0128 (TAK-228) dosing will be adjusted 
accordingly. 
 
In Cycle 1, MLN1028 (TAK-228) and ziv-aflibercept will not be given on the same day. In 
Cycle 2 and beyond, every effort will be made so that MLN0128 (TAK-228) and ziv-
aflibercept will not be given on the same day.  The starting dose is Dose Level 1, which is 
MLN0128 (TAK-228) 4 mg oral for 3 days on and 4 days off and ziv-aflibercept 2 mg/kg 
every 2 weeks.  If Dose Level 1 is beyond the MTD, the dose will be de-escalated to Dose 
Level -1.  If Dose Level -1 is beyond the MTD, then the PI will discuss with NCI-CTEP. 
 
Patients will be restaged at the end of every two cycles. Patients may be allowed to continue 
treatment after 2 cycles if there is continued clinical response or disease stabilization, and 
patients do not have significant toxicities. There is no maximum number of cycles a patient 
may receive if the patient is benefitting clinically. 
 
Dose Escalation Schedule 
Dose Level 
Dose* 
MLN0128 (TAK-228) 
(mg PO for 3 days on and 4 days off) 
Starting on Cycle 1 Day 2 
Ziv-Aflibercept 
(mg/kg IV Q 2 weeks) 
Starting on Cycle 1 Day 1 
Level -1 
3 
2 
Level 1 
4 
2 
Level 2 
4 
3 
*Doses are stated as exact dose in units (e.g., mg/m2, mcg/kg, etc.) rather than as a percentage. 
As of Amendment 13/ Version 07, all newly enrolled patients will be given the milled formulation of MLN0128 
(TAK-228), and a new dose escalation is being explored. Starting with Amendment 14/Version 08, the dose 
escalation will be re-started at Dose Level 1 with the updated dose level escalation. 
Both study drugs will have a +/- 2 day window. If the ziv-aflibercept dosing is rescheduled, then the MLN0128 
(TAK-228) dosing will be adjusted accordingly. 
 
 
 
Regimen Description 

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37 
 
Agent 
Premedications; Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
MLN0128 
(TAK-228) 
Patient should refrain from eating 
2 hours before and 1 hour after. 
May take with 5- HT3 antagonist 
for antinausea 30 min. prior to 
dosing 
(see 
Section 
5.3 
Guidelines for Supportive Care) 
**Gelatin 
capsule 
PO in the AM 
3 Days on and 4 
Days off, Starting 
on Day 2 of 
every cycle 
 
28 days  
(4 weeks) 
Ziv- 
Aflibercept 
No premedications needed. *** 
Need 0.2 micron 
polyethersulfone filter (PVC 
with DEHP or polyurethane) 
** in NS or 
D5W 
Conc. Between 
0.6 – 8 mg/mL 
IV over 60 
minutes 
Days 1 and 15 
**Doses as appropriate for assigned dose level. 
***May give premeds in subsequent doses after 1st dose if infusion related side effects (premeds may consist of 
diphenhydramine, steroids) 
 
The patient will be requested to maintain a medication diary of each dose of medication 
(Appendix D).  The medication diary will be returned to clinic staff at the end of each course. 
 
MLN0128 (TAK-228) will be administered on an empty stomach.  Patients should be 
instructed to refrain from eating and drinking (except for water and prescribed medications) 
for 2 hours before and 1 hour after each dose.   
 
Patients should be instructed to take their study medication at approximately the same time 
on each scheduled dosing day and not to take more than the prescribed dose at any time.  
Patients should swallow the study medication whole and not chew it, open it, or manipulate it 
in any way before swallowing.  If a patient does not take their MLN0128 (TAK-228) dose 
within the time frame specified (±24 hours of the QW scheduled dosing time, or +/- 12 hours 
of the QD×3 days per week schedule), then the dose should be skipped and considered a 
missed dose.  Patients should record any missed doses in their diary and resume drug 
administration at the next scheduled time with the prescribed dosage.  Under no 
circumstance should a patient repeat a dose or double-up doses. 
 
5.1.1 MLN0128 (TAK-228) 
 
MLN0128 (TAK-228) drug product will be supplied in the form of gelatin capsules and taken 
orally by mouth at approximately the same time every morning. Most commonly reported drug- 
related AEs included diarrhea (24%), fatigue (23%), vomiting (19%), rash (19%), dehydration 
(19%), hyperglycemia (19%), mucosal inflammation (16%), asthenia (15%), dysgeusia (13%), 
thrombocytopenia (11%), stomatitis (10%) and blood creatinine increased (8%). 
 
Patients should take MLN0128 (TAK-228) with 8 ounces of water and should refrain from 
eating (except water and prescribed medications) for 2 hours before and 1 hour after each dose.   
Patients may also use 5-HT3 antagonist such as ondansetron, phenothiazines such as 
promethazine or prochlorperazine, benzodiazepines such as lorazepam as premedications for 
MLN0128 (TAK-228) per supportive care guideline under Section 5.3.  Patients will be 
counseled to take medication with full glass of water and to keep well hydrated during the day 

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to prevent hyperglycemic episodes and prevent renal impairment. MLN0128 (TAK-228) is 
metabolized primarily via CYP1A2, with minor contribution from CYP3A4. Strong CYP1A2 
inhibitors and CYP inducers should be administered with caution and at the discretion of the 
investigator during the study. 
 
In cases where a subject misses dosing at his/her dosing time, the subject may still take the dose 
within 12 hours of the regular dosing time (subjects should not take 2 consecutive daily doses 
within 12 hours of each other). Subjects who vomit after receiving MLN0128 (TAK-228) will 
not receive a replacement dose within 24 hours.  If confirmed that the study drug has been 
vomited, the dose should be noted as having been missed. 
 
If severe emesis or mucositis prevents the patient from taking scheduled doses, that dose will be 
skipped. If emesis occurs after study medication ingestion, the dose will not be readministered, 
and patients should resume dosing at the next scheduled time with the prescribed dosage. 
Patients should record the occurrence of the emesis in their dosing diaries. Under no 
circumstance should a patient repeat a dose or double-up doses. 
 
 
5.1.2 Ziv-Aflibercept 
 
Ziv-Aflibercept solution for injection is available as a 200 mg vial (25 mg/mL).  It is compatible 
in either 0.9% sodium chloride or 5% dextrose solutions and is not to be mixed with other drugs. 
Ziv-Aflibercept solutions at 0.6 to 8 mg/mL are stable for up to 24 hours under refrigerated 
conditions (2 to 8°C) or up to 8 hours at ambient temperature (approximately 25°C) in 
polypropylene syringes or in infusion bags made of polyvinyl chloride (PVC) containing di(2-
ethylhexyl)phthalate (DEHP) or polyolefin (PVC free DEHP free). Diluted solutions of ziv-
aflibercept should be administered using infusion tubing made of PVC containing DEHP, DEHP 
free PVC containing tris(2-ethylhexyl) trimellitate (TOTM), polypropylene, polyethylene lined 
PVC, or polyurethane. The infusion sets must contain a 0.2 μm polyethersulfone inline filter. 
Polyvinylidene fluoride (PVDF) filters and Nylon filters should not be used. Infusion can be 
conducted by gravity, with an IV infusion pump, or with a syringe pump using administration 
sets made of the above materials. The infusion should not exceed two hours at room temperature 
(approximately 25°C). 
 
The most common treatment-emergent adverse events reported on IV VEGF Trap treatment 
were fatigue, pain, and constipation.  Potentially drug-related severe adverse events encountered 
to date have included hypertension, fatigue, dyspnea, nausea/vomiting, headache, arthralgia, 
hoarseness, and lower extremity deep vein thrombosis. 
 
There are no premedications recommended for ziv-aflibercept. However, if patients experience 
first dose infusion related side effects, subsequent doses may be given with antihistamines, H2 
antagonists, and steroids, per institutional standards for drug infusion reactions. 
 
 
 
 
5.2 
Definition of Dose-Limiting Toxicity 

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Management and dose modifications associated with the above adverse events are outlined in 
Section 6. 
 
Dose escalation will proceed within each cohort according to the following scheme.  Dose- 
limiting toxicity (DLT) is defined below. 
 
Number of Patients with DLT at a Given 
Dose Level 
Escalation Decision Rule 
0 out of 3 
Enter 3 patients at the next dose level. 
>2 
Dose escalation will be stopped. This dose level will be declared 
the maximally administered dose (highest dose administered). 
Three (3) additional patients will be entered at the next lowest dose 
level if only 3 patients were treated previously at that dose. 
1 out of 3 
Enter at least 3 more patients at this dose level. 
• If 0 of these 3 patients experience DLT, proceed to the next dose 
level. 
• If 1 or more of this group suffer DLT, then dose escalation is 
stopped, and this dose is declared the maximally administered 
dose. Three (3) additional patients will be entered at the next 
lowest dose level if only 3 patients were treated previously at 
that dose. 
≤1 out of 6 at highest dose level below the 
maximally administered dose 
This is generally the recommended phase 2 dose. At least 6 patients 
must be entered at the recommended phase 2 dose. 
 
Definition of DLT 
 
CTCAE version 5.0 will be utilized. DLTs will be based on events occurring during the first 
cycle of study drug administration (DLT window is 1 cycle= 28 days).  If multiple toxicities are 
seen, the presence of DLT shall be based on the most severe toxicity experienced.  
 
Patients who experience an AE that meets the definition of a DLT during or after completing 
Cycle 1 should have their study drug treatment interrupted.  If the event resolves to grade 1 or 
baseline values within 3 weeks of interrupting planned therapy, and in the opinion of the 
investigator the benefits of continuing treatment outweigh the risks posed by the toxicity, 
patients may continue study treatment with MLN0128 (TAK-228) dose reduction. 
 
DLTs are defined as adverse events (AEs) related to study agents with an attribution of possible, 
probably, or definite and fulfilling one of the following criteria: 
 
Hematologic 
• Grade 4 neutropenia lasting > 7 days 
• Febrile neutropenia (defined as absolute neutrophil count [ANC] < 1.0 x 109/L and 
fever ≥ 38.5o C) or documented grade ≥ 3 infection with ANC ≤ 1.0 x 109/L. 
• Platelet count < 25,000/mm lasting > 7 days 

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40 
 
• Delay of treatment > 7 days due to hematologic toxicity 
 
Non-hematologic 
• Any toxicity ≥ grade 3 that persists for > 7 days, except: 
1. nausea/vomiting, diarrhea and electrolyte imbalances; 
2. grade 3 lab abnormalities that are asymptomatic and responsive to supportive 
measures and that are without clinical consequence; and 
3. grade 3 hyperglycemia or grade 3 diabetes that can be stably controlled. 
• Delay of treatment > 14 days due to non-hematologic toxicity 
 
EXCEPTION: 
Patients who experience grade 3 hypertension that resolves within 14 days with medical 
management will not be defined as a DLT. 
 
Patients who experience proteinuria that resolves to <2 g within 14 days will not be defined as 
a DLT. 
 
Hypersensitivity/Allergic reactions with expected severity and presentation will not be 
considered a DLT 
 
Patients who withdraw from the study prior to completion of the first cycle of study treatments 
for reasons other than treatment-related adverse events will be replaced. 
 
Patients can have treatment withheld for up to 1 cycle (28 days) for reasons other than toxicity 
(i.e. social reasons). 
 
 
5.3 
General Concomitant Medication and Supportive Care Guidelines 
 
Subjects must not receive any other investigational drugs or any other antineoplastic therapy 
including radiotherapy while participating in this study. Supportive treatment such as 
antiemetics, antidiarrheals, analgesics, blood products, etc., may be used at the investigator’s 
discretion. Concurrent palliative radiation may be permitted after discussing with CTEP. 
 
5.3.1 Concomitant Medications 
 
The PI or pharmacist or PI delegate will go over and check the list of medication for individual 
patients. 
 
Because there is a potential for interaction of MLN0128 (TAK-228) with other concomitantly 
administered drugs through the cytochrome P450 system, the case report form must capture the 
concurrent use of all other drugs, over-the-counter medications, or alternative therapies.  The 
Principal Investigator should be alerted if the patient is taking any agent known to affect or with 
the potential to affect selected CYP450 isoenzymes.  Appendix C presents guidelines for 
identifying medications/substances that could potentially interact with the study agent(s). 
 

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41 
 
Strong CYP1A2 inhibitors and CYP inducers should be administered with caution, at the 
discretion of the investigator. Alternative treatments, if available, should be considered. 
Histamine H2 receptor antagonists may be allowed, if needed provided that the histamine H2 
receptor antagonist is not taken within 12 hours before and within 6 hours after study drug 
administration. Patients receiving histamine H2 receptor antagonists before enrollment must stop 
using these medications for at least 24 hours before their first dose of study drug. Examples of 
histamine H2 receptor antagonists include ranitidine, famotidine, and nizatidine. Cimetidine, a 
moderate cytochrome P450 (CYP)1A2 inhibitor, is not recommended as a first choice H2 
receptor antagonist. Strong CYP1A2 inhibitors and CYP inducers should be administered with 
caution, at the discretion of the investigator (see Appendix C). Alternative treatments, if 
available, should be considered. 
 
The use of live vaccines and close contact with those who have received live vaccines should be 
avoided during treatment with study drug. Examples of live vaccines are: intranasal influenza, 
measles, mumps, rubella, oral polio, Bacille Calmette-Guerin, yellow fever, varicella, and TY21a 
typhoid vaccines. 
 
Systemic corticosteroids (either IV or oral steroids, excluding inhalers) should be excluded 
unless necessary for treatment a TAK-228-related AE (eg, rash) as recommended by the treating 
physician. 
 
Neutralizing antacid preparations (acid neutralizers) and calcium supplements are not permitted 
during Cycle 1 on study drug administration days in the phase 1/1b portion of the study, but may 
be taken as needed on non-TAK-228 administration days. However, for all other cycles, 
administration of neutralizing antacids and calcium preparations is permitted except from 4 hours 
before until 2 hours after TAK-228 administration. Some anti-gas preparations may also have 
antacid properties, and should also not be permitted from 4 hours before until 2 hours after study 
drug administration. 
 
5.3.2 Supportive Care 
• Hypersensitivity reactions with any dose: Stop infusion of ziv-aflibercept for 30-60 
minutes. Medicate with diphenhydramine 25 – 50 mg IV (or a similar antihistamine) and 
steroids such as hydrocortisone 50-100 mg IV or dexamethasone 10-20 mg IV 
approximately 30 minutes before re- starting the ziv-aflibercept.  If the subject re- 
develops a hypersensitivity reaction despite treatment with diphenhydramine and 
steroids, the infusion should be stopped for another 30 – 60 minutes, depending upon the 
severity of the reaction.  The infusion may be resumed by administering a histamine H2- 
receptor antagonist approximately 30 minutes before restarting the ziv-aflibercept 
infusion and meperidine 25-50 mg IV if patient still had chills and rigors.  Famotidine 20 
mg IV or ranitidine 50 mg IV are recommended rather than cimetidine because of the 
lack of likely metabolic/pharmacologic interactions with the former drugs.  The rate of 
the ziv-aflibercept infusion may also be slowed from 60 minutes to 2 hours. Also, it is 
permissible to premedicate overnight with steroids such as dexamethasone or prednisone. 
All subjects should be monitored while receiving the ziv-aflibercept infusion and 
emergency medical equipment and health care personnel must be readily available to 
respond to hypersensitivity reactions or other medical emergencies. 

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• Nausea/vomiting induced by study agents should be treated with a serotonin receptor 
antagonist (5HT3) ondansetron 8 mg PO 30 min. prior to dosing then Q 8 hours prn, or 
using phenothiazine derivatives such as prochlorperazine 10 mg PO Q 8 hours prn or 
promethazine 25 mg PO Q 6 hours prn as breakthrough.  If this is inadequate, a 
benzodiazepine such as lorazepam or corticosteroid such as dexamethasone (except with 
MLN0128 (TAK-228)) or prednisone should be added until acute nausea is controlled.  
After acute nausea has resolved, consideration should be given to initiation of 
prophylactic antiemetic therapy.  If nausea recurs despite reasonable medical 
intervention (as outlined above), dose reduction will be needed. 
 
• Diarrhea should be managed with loperamide:  4 mg at first onset, then 2 mg every 2-4 
hours until diarrhea is controlled (maximum = 16 mg loperamide/day). 
Diphenoxylate/Atropine (Lomotil™) may be added and alternate with loperamide. 
 
• Hyperlipidemia may be treated with appropriate lipid lowering agents. 
 
• Management of Hyperglycemia 
On the basis of the clinical experience in TAK-228 trials, most episodes of 
hyperglycemia observed occurred within the first 60 days after initiation of treatment 
with TAK-228 and have been either Grade 1 or Grade 2, and have responded quickly to 
oral metformin. Hyperglycemia has not been dose-limiting since the institution of a 
standard regimen for early treatment of hyperglycemia.  
 
All patients developing hyperglycemia during the study should have their glucose closely 
monitored by study staff and will be referred to our endocrinologist collaborator for 
management. The investigator may choose to continue close monitoring of patients who 
develop Grade 1 hyperglycemia (fasting glucose >ULN ≤160 mg/dL) or, alternatively, 
consider initiating treatment with an oral hypoglycemic agent, such as metformin. All 
patients with ≥Grade 2 hyperglycemia (fasting glucose >160 mg/dL) must be treated 
aggressively with oral hypoglycemic agents and/or insulin as clinically indicated. The 
investigator should consult an endocrinologist, if needed, to aid in optimizing the 
patient’s hyperglycemia treatment plan. 
 
It is recommended that patients with elevated fasting blood glucose be initially treated 
with a fast acting insulin sensitizer such as metformin at 500 mg orally QD, and titrate up 
to a maximum of 1000 mg orally BID as needed. Concurrent addition to metformin of 
DPP-4 inhibitors (eg, sitagliptin or vildagliptin) and/or insulin should also be considered. 
Oral sulfonylureas (eg, glipizide or glyburide) should be used with caution, due to the 
higher risk of inducing hypoglycemia in patients. The dose of oral hypoglycemic agents 
should be adjusted in patients with renal insufficiency. In addition, patients should be 
encouraged to follow a low carbohydrate diet once hyperglycemia is first observed. 
If any fasting serum glucose reading performed at the site indicates hyperglycemia 
(>ULN or ≥110 mg/dL), the study staff should first confirm that the patient was fasting at 
the time of blood specimen collection (ie, nothing by mouth for at least 8 hours before 
collection). 

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• In-Home Daily Fasting Blood Glucose (FBG) Monitoring 
In addition to obtaining fasting glucose levels at the clinic visits as outlined in the 
Schedule of Events, all patients will be given a glucometer to monitor their daily FBG 
levels at home. The level should be collected daily, predose on dosing days, and at 
approximately the same time each day. 
 
On Cycle 1 Day 1, the patient will be provided an in-home glucometer. Patients should 
be trained on proper use of the glucometer and instructed to collect a daily FBG level 
every morning (predose on dosing days), starting on Cycle 1 Day 2. Patients will be 
instructed to bring the glucometer with them to each study visit so that the data collected 
can be reviewed and recorded in the source documents (Appendix E). Investigators will 
be responsible for reviewing the home glucose monitoring logs for hyperglycemia. 
The patient will be instructed to contact the site immediately if the value is abnormal (ie, 
≥150 mg/dL) for further instructions on the management of their hyperglycemia. 
Hyperglycemia observed during home glucose monitoring should be confirmed in the 
clinic. 
 
If no irregularities in the fasting blood glucose level are observed during a minimum of 2 
consecutive months, then the frequency of in-home fasting blood glucose testing can be 
reduced to a minimum frequency of once weekly, depending on the investigator’s 
judgment and approval. Patients will continue to notify the investigator of fasting blood 
glucose levels that exceed 150 mg/dL and, if blood glucose levels are not well controlled, 
or if the patient requires either oral hypoglycemic agents or insulin to control blood 
glucose levels, then the frequency of in-home testing of FBG levels will be reinstated to 
daily. 
 
• Suggested management of mucositis: 
• Xyloxylin (1:1:1 ratio) (diphenhydramine, Maalox, lidocaine) 10 mL 
swish/swallow QID as needed 
• Caphosol (sodium phosphate) 15 mL swish/spit every 4 hours as needed 
Valacyclovir 500 mg po TID for treatment 
• Biotene MW every 4 hours as needed 
• Carafate 1 Gm/10 mL 10 mL swish/swallow or spit QID as needed 
• Glutamine swish/swallow BID as needed (1 scoop (4 grams) mixed with water) 
 
• Routine supportive measures for cancer patients such as erythropoietin, analgesics, blood 
transfusions, antibiotics, bisphosphonates, and hematopoietic colony stimulating factors 
for treatment of cytopenias are permitted. 
 
• The administration of anticancer therapies, other investigational agents, or prophylactic 
use of hematopoietic colony stimulating factors are not permitted during Cycle 1; 
however, Hematopoietic colony stimulating factors may be used in subsequent cycles per 
guidelines and PI discretion. Accepted standards such as the American Society of 
Clinical Oncology (ASCO) Guidelines on the Use of Hematopoietic Colony-Stimulating 
Factors should be followed. 

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5.4 
Duration of Therapy 
 
In the absence of treatment delays due to adverse event(s), treatment may continue until one 
of the following criteria applies: 
 
• Disease progression per RECIST criteria, 
 
• Intercurrent illness that prevents further administration of treatment, 
 
• Unacceptable adverse event(s), 
 
• Patient decides to withdraw from the study, or 
 
• General or specific changes in the patient's condition render the patient 
unacceptable for further treatment in the judgment of the investigator. 
 
5.5 
Duration of Follow Up 
 
Patients will be followed for 4 weeks (+/- 7 days) after removal from study or until death, 
whichever occurs first.  Patients removed from study for unacceptable adverse event(s) will be 
followed until resolution or stabilization of the adverse event. Every attempt will be made to 
adhere to 4 weeks; however, the follow-up visit will depend on the patient’s ability to return 
and at the physician’s discretion. 
 
5.6 
Criteria for Removal from Study 
 
Patients will be removed from study when any of the criteria listed in Section 5.4 applies. The 
reason for study removal and the date the patient was removed must be documented in the 
Case Report Form. 

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45 
 
6. DOSING DELAYS/DOSE MODIFICATIONS 
 
Subjects will be carefully instructed to notify the Principle Investigator or study 
coordinator at the first appearance of any toxicity.  The study coordinator will speak with 
subjects on clinic visit days to determine the degree and duration of any toxicity.  Toxicity 
will be evaluated using the CTCAE version 5.0. Subject data will be analyzed for 
evidence of cumulative toxicity with repeated courses of therapy. 
 
Please note, per the physician’s discretion, a patient may continue MLN0128 (TAK-228) 
alone if the patient is receiving clinical benefit, but has to stop ziv-aflibercept for toxicity 
reasons. 
 
Dose modifications for toxicity (in individual patients) relative to MLN0128 (TAK-228) 
in Dose Level 1 and 2: 
 
MLN0128 (TAK-228) 
(mg PO 3 days on and 4 days off starting Day 2 of 
every Cycle) 
3 mg 
Discontinue 
 
 
If patients in MLN0128 (TAK-228) Dose Level -1 require dose modification, those patients 
will have drug discontinuation. 
 
 
 
 
 
 
 
 
Dose modifications for toxicity (in individual patients) relative to ziv-aflibercept in Dose 
Level 3 through Dose Level 1: 
 
Ziv-Aflibercept 
(mg/kg IV Q 2 weeks) 
2 
Discontinue 
 
If patients in ziv-aflibercept Dose Level -1 require dose modification, those patients will 
have drug discontinuation. 
 
Intra-patient dose (re-)escalation (after toxicity induced reduction) will be allowed on a case-by- 
case basis and after discussion and approval by CTEP. 
 
Treatment Modification (in individual patients): 
 
Nausea/Vomiting 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 

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46 
 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
No change in dose. Maximize anti-
emetic therapy. Consider IV fluid 
hydration.  
Hold until ≤ Grade 1. Resume at same 
dose level. 
Grade 3 
Maximize anti-emetic therapy. 
Initiate tube feeding, IVF or TPN.  
If experienced for ≤72 hours, hold 
TAK-228 until ≤Grade 1, then 
resume TAK-228 without dose 
modification. If experienced for >72 
hours despite optimal therapy, hold 
TAK-228 until ≤ Grade 1, then 
resume treatment with the dose of 
TAK-228 reduced by 1 level.  
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Grade 4 
Maximize anti-emetic therapy. 
Initiate tube feeding, IVF or TPN.  
If experienced for ≤72 hours, hold TAK-
228 until ≤Grade 1, then resume TAK-228 
without dose modification. If experienced 
for >72 hours despite optimal therapy, hold 
TAK-228 until ≤ Grade 1, then resume 
treatment with the dose of TAK-228 
reduced by 1 level. 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should discontinue ziv-aflibercept, but may continue MLN0128 (TAK-228). 
**Patients requiring > two dose reductions should go off protocol therapy. 
Prevention/Prophylaxis: 
Prophylactic use of anti-emetic, antinausea, and antidiarrheal medications are encouraged and may be used before 
each TAK-228 dosing as needed throughout the study. 
IV=intravenous, IVF=intravenous fluids, TPN=total parental nutrition. 
 
 
 
 
 
Diarrhea 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
Hold until ≤ Grade 1. Resume at same 
dose level. 
Hold until ≤ Grade 1. Resume at same 
dose level. 
Grade 3 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Grade 4 
Off protocol therapy 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
Recommended management: Loperamide antidiarrheal therapy 
Dosage schedule: 4 mg at first onset, followed by 2 mg with each loose motion until diarrhea-free for 12 hours 
(maximum dosage: 16 mg/24 hours) 
Adjunct anti-diarrheal therapy is permitted and should be recorded when used. 
 
 
Neutropenia 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 

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47 
 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
Hold until ≤ Grade 1. Resume at same 
dose level. 
Hold until ≤ Grade 1. Resume at same 
dose level. 
Grade 3 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Grade 4 
Off protocol therapy 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
 
 
Thrombocytopenia 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
Hold until ≤ Grade 1. Resume at same 
dose level. 
Hold until ≤ Grade 1. Resume at same 
dose level. 
Grade 3 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Grade 4 
Off protocol therapy 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
 
 
Mucositis/ Stomatitis 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
Hold until ≤ Grade 1. Resume at one 
dose level lower. 
Hold until ≤ Grade 1. Resume at one 
dose level lower. 
Grade 3* 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Hold* until < Grade 2. Resume at one 
dose level lower, if indicated.** 
Grade 4* 
Off protocol therapy 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
Recommended management: See Section 5.3 for supportive care and suggested management of mucositis. 
 
 
Hypertension 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose*** 
No change in dose*** 
Grade 2 
No change in dose*** 
No change in dose*** 
Grade 3* 
If hypertension is controlled within 2 
weeks of the delay, resume MLN0128 
(TAK-228) at same dose level. 
Delay the administration of ziv- 
aflibercept for a maximum of 2 weeks, 
until recovery to blood pressure (BP) ≤ 
150/100 or to systolic BP < 180 if 
diastolic BP < 90 for patients with known 
history of isolated systolic hypertension: 
•If BP is controlled within 2 weeks delay: 
-First episode: re-administer ziv- 
aflibercept at one dose level 
lower. 
- Second episode: re-administer ziv- 
aflibercept with reduced to two dose 
levels lower. 
-Third episode, discontinue ziv- 

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aflibercept. 
•In case of re-occurrence of grade 3 BP 
despite dose reduction of ziv-aflibercept, 
or if BP is still uncontrolled despite 1 
omission of administration of ziv- 
aflibercept, the patients will be 
permanently discontinued from ziv- 
aflibercept. MLN0128 (TAK-228) will be 
continued if the investigator thinks the 
patient is benefiting from it. 
Grade 4* 
If hypertension is controlled within 2 
weeks of the delay, resume MLN0128 
(TAK-228) at same dose level. 
Seek cardiologist opinion, and 
permanently discontinue. 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
***Initiate antihypertensive drug therapy and close monitoring of blood pressure, as needed. 
 
 
Proteinuria 
Management/Next Dose for 
MLN0128 (TAK-
228) 
Management/Next Dose for Ziv-Aflibercept 
UPCR*** [0-1] 
No change in dose 
No change in dose 
UPCR [1-2] 
Absence of Hematuria 
No change in dose 
Perform 24h urine: 
• 
If ≤3.5g/24h: 
o 
Resume at one dose level lower 
o 
>2g/24h prior current dosing then 
omit dosing and repeat 24h urine, 
if: 
 
≤2g/24h prior current 
dosing, resume at one 
dose level lower 
 
>2g/24h prior current 
dosing, discontinue 
• 
If >3.5g/24h and previous 24h urine was: 
o 
≤2g/24h prior current dosing then 
resume at one dose level lower 
o 
>2 ≤ 3.5g/24h prior current dosing 
then omit dosing and repeat 24h 
urine, if: 
 
≤2g/24h prior current 
dosing, resume at one 
dose level lower 
 
>2g/24h prior current 
dosing, discontinue 
o 
>3.5g/24h prior current dosing 
then discontinue 
UPCR [1-2] Presence 
of Hematuria OR 
UPCR >2 
No change in dose 
Omit dosing ziv-aflibercept then perform 24h 
urine 
• 
Full nephrologic work-up and seek 
nephrologist opinion 
• 
TMA*** not suspected and ≤2g/24h 
prior current dosing 
o 
Dose ziv-aflibercept 

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Proteinuria 
Management/Next Dose for 
MLN0128 (TAK-
228) 
Management/Next Dose for Ziv-Aflibercept 
o 
If second occurrence, resume at 
one dose level lower 
• 
TMA not suspected and >2 ≤ 3.5g/24h 
prior current dosing,  omit dosing and 
perform 24h urine, if: 
o 
≤2g/24h prior current dosing, 
then resume at one dose level 
lower 
o 
>2g/24h prior current dosing, 
then discontinue 
• 
TMA not suspected and >3.5g/24h 
prior current dosing****, 
discontinue 
• 
TMA diagnosed, discontinue and seek 
nephrologist opinion 
Nephrotic Syndrome 
If proteinuria is controlled within 2 
weeks of the delay, resume MLN0128 
(TAK-228) at same dose level 
Off protocol therapy 
 Note: Patients requiring a delay of >2 weeks should discontinue ziv-aflibercept, but may continue MLN0128 (TAK-
228). Patients requiring > two dose reductions should go off protocol therapy. 
***UPCR: Urinary protein creatinine ratio; TMA: Thrombotic micro-angiopathy 
****When patient is already treated at lowest dose level, ziv-aflibercept should be discontinued. 
 
 
Hyperglycemia 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose; Continue close 
monitoring of blood sugars. Initiate oral 
hypoglycemic agent. 
No change in dose 
Grade 2 
No change in dose; Initiate oral 
hypoglycemic agent and/or insulin if not 
well controlled on oral agent 
No change in dose 
 
Grade 3 
Hold drug until ≤ Grade 2. 
Resume MLN0128 (TAK-228) based 
on timing of recovery: 
≤ 1 week: resume at same dose and 
schedule; 
>1 but ≤ 2 weeks: reduce TAK-228 by 
1 dose level 
> 2 weeks: stop MLN0128 (TAK-
228) and discontinue subject from 
the study. 
No change in dose. 
Grade 4 
Off protocol therapy 
If hyperglycemia is controlled within 2 
weeks of the delay, resume ziv-aflibercept 
at same dose level 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
Note: The investigator will work closely with the endocrinologist, and metformin will be the recommended oral 
hyperglycemic agent. 
Prevention/Prophylaxis: 

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• 
Follow fasting glucose levels during clinic visits. 
• 
Monitor home glucometer test results. 
• 
Check HbA1c levels every 3 months during therapy. 
• 
Recommend life-style modifications, as appropriate (balanced diet, limited alcohol consumption, increased 
physical activity). 
• 
Most episodes of Grade 1 or 2 hyperglycemia respond quickly to oral metformin. Early initiation of therapy 
at the lowest therapeutic dose is recommended to prevent higher grade hyperglycemia. 
• 
Fasting blood glucose levels ≥150 mg/dL by glucometer should be followed by closer monitoring of serum 
glucose and possible intervention. 
 
 
 
Pneumonitis and 
Non-Infectious 
Pneumonitis 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose; Rule out infection and 
closely monitor 
No change in dose 
Grade 2 
Rule out infection and consider treatment 
with corticosteroids until symptoms 
improve to ≤ Grade 1. 
Interrupt MLN0128 (TAK-228) treatment: 
When symptoms ≤ Grade 1, re-initiate 
MLN0128 (TAK-228) treatment at a Dose 
reduced by 1 level. If no recovery within 4 
weeks, then discontinue MLN0128 (TAK-
228). 
No change in dose. 
Grade 3 
Rule out infection and consider treatment 
with corticosteroids until symptoms 
improve to ≤ Grade 1. 
Interrupt MLN0128 (TAK-228) 
treatment until symptoms 
resolve to ≤ Grade 1. 
At the physician’s discretion, 
consider re-initiating MLN0128 
(TAK-228) treatment at a dose 
reduced by 1 level. If toxicity 
recurs at Grade 3, discontinue 
MLN0128 (TAK-228) 
treatment. Obtain PFTs and 
pulmonary consultation. 
If pneumonitis improves within 2 weeks 
of the delay, resume ziv-aflibercept at 
same dose level.** 
Grade 4 
Off protocol therapy 
If pneumonitis improves within 2 weeks 
of the delay, resume ziv-aflibercept at 
same dose level.** 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
 
 
Hyperlipidemia 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
Treat hyperlipidemia according to 
standard guidelines.- Maintain dose if 
No change in dose 

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tolerable 
Triglycerides ≥ 500 mg/dl should be 
treated urgently due to risk of pancreatitis- 
If toxicity becomes intolerable, interrupt 
MLN0128 (TAK-228) dosing until 
recovery to ≤ Grade 1. Reinitiate at same 
dose. 
Grade 3 
Treat hyperlipidemia according to 
standard guidelines. Hold* until < Grade 
2. Resume at one dose level lower, if 
indicated.** 
If hyperlipidemia is controlled within 2 
weeks of the delay, resume ziv-aflibercept 
at same dose level 
Grade 4 
Off protocol therapy 
If hyperlipidemia is controlled within 2 
weeks of the delay, resume ziv-aflibercept 
at same dose level 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
 
 
Rash 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
No change in dose. Symptomatic 
treatment. Consider treatment 
with topical steroid 
cream/ointment and/or oral anti-
histamines or antibiotics. 
No change in dose 
Grade 3 
Dermatology consultation. Consider 
treatment with topical steroid 
cream/ointment and/or oral anti-
histamines or antibiotics. Hold* until 
</ = Grade 2. Resume MLN0128 (TAK-
228) based on timing of recovery: 
≤ 3 weeks: Reduce TAK-228 by 1 dose 
level; 
> 3 weeks: stop MLN0128 (TAK-
228) and discontinue subject from 
the study 
If rash improves within 2 weeks of the 
delay, resume ziv-aflibercept at same dose 
level.** 
Grade 4 
Patients who develop Grade 4 rash should 
permanently discontinue study treatment, 
unless they derive clinical benefit, in 
which case they may be retreated at a 
reduced dose level after recover to ≤ 
Grade 1 severity. Grade 4 rash is defined 
as rash acneifom/papulopustular with 
papules and/or pustules covering any % 
body surface area, which may or may not 
be associated with symptoms of pruritus 
or tenderness, and are associated with 
extensive superinfection with intravenous 
(IV) antibiotics indicated; life threatening 
consequences (NCI CTCAE Version 5.0). 
 
If rash improves within 2 weeks of the 
delay, resume ziv-aflibercept at same dose 
level.** 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 

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Rash 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
**Patients requiring > two dose reductions should go off protocol therapy. 
Prevention/Prophylaxis: 
• 
Rash should be managed aggressively. The investigator should consider consulting a dermatologist or other 
specialist, if needed. 
• 
A skin biopsy at the site of rash should be considered as soon as possible after the initial episode. 
 
 
QTc Prolongation 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose 
No change in dose 
Grade 2 
Evaluate for other possible causes (eg, 
electrolyte disturbance, concomitant 
medication, etc.) 
Continue MLN0128 (TAK-228) at the 
same dose and schedule. 
No change in dose. 
Grade >/=3* 
Evaluate for other possible causes (eg, 
electrolyte disturbance, concomitant 
medication)a; 
Consider a formal consult by a 
cardiologist; Notify the study doctor; 
Additional ECGs may be performed 
at intervals that the treating physician 
deems clinically appropriate until 
repeated QTc measurements fall or 
are below the threshold interval that 
triggered the repeat measurement. 
Notify the study doctor; 
Additional ECGs may be performed 
at intervals that the treating physician 
deems clinically appropriate until 
repeated QTc measurements fall or 
are below the threshold interval that 
triggered the repeat measurement.  
 
Hold TAK-228 
Patients who experience persistent 
symptomatic Grade 3 or Grade 4 QTc 
prolongation without another cause 
should permanently discontinue study 
treatment.  
 
If QTc prolongation improves within 2 
weeks of the delay, resume ziv-aflibercept at 
same dose level.** 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy. 
ECG=electrocardiogram, IV=intravenous, msec=milliseconds, QTc=QT interval corrected for heart rate. 
(a) A list of medications known to prolong QTc can be found at https://www.crediblemeds.org/new-drug-list/  
 
 
 
Other Non-Hematologic 
Toxicities (Including 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 

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53 
 
Asthenia, Weakness, and 
Fatigue) 
≤ Grade 1 
Initiate appropriate medical therapy and 
monitor. 
If tolerable, then no adjustment is required. 
No change in dose 
Grade 2 
Initiate appropriate medical therapy and 
monitor. If tolerable, no adjustment 
required. 
If toxicity becomes intolerable, hold 
TAK-228 until recovery to ≤Grade 1, 
then reinitiate at same dose. 
No change in dose 
Grade >/=3* 
Hold TAK-228 until recovery to 
≤ Grade 1. Reinitiate TAK-228 at dose 
reduced by 1 level  
Patients who develop Grade 4 
nonhematological toxicities (with the 
exception of isolated non-clinically 
significant laboratory values) should 
permanently discontinue study treatment, 
unless they derive clinical benefit, in 
which case they may be retreated at a 
reduced dose level after recovery to ≤ 
Grade 1 severity. 
 
No change in dose. 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
 
 
 
Aspartate 
Aminotransferase/ 
Alanine 
Aminotransferase 
Elevations 
Management/Next Dose for MLN0128 
(TAK-228) 
Management/Next Dose for Ziv- 
Aflibercept 
≤ Grade 1 
No change in dose. 
No change in dose 
Grade 2 
No change in dose;  
Closely monitor LFTs at least weekly or 
more frequently as indicated. 
Assess patient for other causes of 
transaminitis (eg, past medical history, 
concomitant medications). 
No change in dose 
Grade 3* 
Closely monitor LFTs at least weekly or 
more frequently as indicated. 
Assess patient for other causes of 
transaminitis (eg, past medical history, 
concomitant medications). 
Hold TAK-228 until ≤Grade 1; 
Restart TAK-228 at the same dose. 
Permanently discontinue study 
treatment if in combination with 
Grade 2 total bilirubin elevation 
when alternative causes cannot be 
identified (ie, Hy’s Law). 
No change in dose. 

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54 
 
Grade 4* 
Closely monitor LFTs at least weekly or 
more frequently as indicated. 
Assess patient for other causes of 
transaminitis (eg, past medical history, 
concomitant medications). 
Stop TAK-228 and discontinue patient 
from the study. Permanently discontinue 
study treatment if in combination with 
Grade 2 total bilirubin elevation when 
alternative causes cannot be identified (ie, 
Hy’s Law). 
No change in dose. 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
Prevention/Prophylaxis: 
Ensure proper screening of patients for study participation. 
LFTs=liver function tests, ULN=upper limit of normal. 
 
 
 
Management of Patients with Possible Cardiac Instability 
 
For patients showing signs of cardiac instability after MLN0128 (TAK-228) administration, 
additional monitoring onsite before clinic discharge should be considered. 
 
 
Management of Specific Adverse Events Related to Ziv-Aflibercept 
 
Discontinue treatment for any of the following: 
• Severe hemorrhage 
• Gastrointestinal perforation 
• Compromised wound healing 
• Fistula formation 
• Hypertensive crisis or hypertensive encephalopathy 
• Arterial thromboembolic events 
• Nephrotic syndrome or thrombotic microangiopathy (TMA) 
• Reversible posterior leukoencephalopathy syndrome. 
 
 
7. ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS 
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. The 
following list of AEs (Section 7.1) and the characteristics of an observed AE (Section 7.2) will 
determine whether the event requires expedited reporting (via CTEP Adverse Event Reporting 
System (CTEP-AERS)) in addition to routine reporting. 
 
7.1 Comprehensive Adverse Events and Potential Risks List(s) (CAEPRs) 
 
The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single list of 
reported and/or potential adverse events (AE) associated with an agent using a uniform 

NCI Protocol #: 9585 
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55 
 
presentation of events by body system.  In addition to the comprehensive list, a subset of AEs, 
the Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column 
and is identified with bold and italicized text.  The SPEER is a list of events that are protocol- 
specific exceptions to expedited reporting to NCI via (CTEP-AERS) (except as noted below). 
Refer to the 'CTEP, NCI Guidelines: Adverse Event Reporting Requirements'  
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm for 
further clarification. 
 
NOTE:  The highest grade currently reported is noted in parentheses next to the AE in the 
SPEER. Report ONLY AEs higher than this grade expeditiously via CTEP-AERS.  If this 
CAEPR is part of a combination protocol using multiple investigational agents and has an AE 
listed on different SPEERs, use the lower of the grades to determine if expedited reporting is 
required. 
 
7.1.1 CAEPRs for CTEP IND Agent(s) 
 
7.1.1.1 
CAEPR for MLN0128 (TAK-228) (NSC# 768435) 
 
Version 2.3, July 28, 20191 
 
 Adverse Events with Possible  
 Relationship to MLN0128 (TAK-228) 
 (CTCAE 5.0 Term) 
[n= 390] 
 
 
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
  
 
Anemia 
 
 Anemia (Gr 2) 
CARDIAC DISORDERS 
  
 
 
Cardiac arrest 
  
 
 
Ventricular fibrillation 
  
GASTROINTESTINAL DISORDERS 
  
 
Abdominal pain 
 
 Abdominal pain (Gr 2) 
Constipation 
 
 
 Constipation (Gr 2) 
Diarrhea 
 
 
 Diarrhea (Gr 2) 
 
Dry mouth 
 
 Dry mouth (Gr 2) 
 
Dyspepsia 
 
  
Mucositis oral 
 
 
 Mucositis oral (Gr 2) 
Nausea 
 
 
 Nausea (Gr 3) 
Vomiting 
 
 
 Vomiting (Gr 3) 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
  
 
Edema limbs 
 
   
Fatigue 
 
 
 Fatigue (Gr 3) 
 
Fever 
 
 Fever (Gr 2) 
 
General disorders and 
administration site 
conditions - Other (mucosal 
inflammation) 
 
 General disorders and 
administration site 
conditions - Other 
(mucosal inflammation) 
(Gr 2) 
INFECTIONS AND INFESTATIONS 
  
 
Urinary tract infection 
 
 Urinary tract infection (Gr 
2) 

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 Adverse Events with Possible  
 Relationship to MLN0128 (TAK-228) 
 (CTCAE 5.0 Term) 
[n= 390] 
 
 
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
 
INVESTIGATIONS 
  
 
Creatinine increased 
 
 Creatinine increased (Gr 2) 
 
 
Electrocardiogram QT 
corrected interval 
prolonged 
  
 
Platelet count decreased 
 
 Platelet count decreased 
(Gr 2) 
 
Weight loss 
 
 Weight loss (Gr 2) 
METABOLISM AND NUTRITION DISORDERS 
  
Anorexia 
 
 
 Anorexia (Gr 2) 
 
Dehydration 
 
 Dehydration (Gr 2) 
Hyperglycemia 
 
 
 Hyperglycemia (Gr 3) 
 
Hypokalemia 
 
 Hypokalemia (Gr 2) 
 
Hypomagnesemia 
 
 Hypomagnesemia (Gr 2) 
 
Hypophosphatemia 
 
 Hypophosphatemia (Gr 2) 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
  
 
Arthralgia 
 
  
 
Back pain 
 
 Back pain (Gr 2) 
 
Pain in extremity 
 
  
NERVOUS SYSTEM DISORDERS 
  
 
Dizziness 
 
 Dizziness (Gr 2) 
 
Dysgeusia 
 
 Dysgeusia (Gr 2) 
 
Headache 
 
 Headache (Gr 2) 
PSYCHIATRIC DISORDERS 
  
 
Insomnia 
 
  
RENAL AND URINARY DISORDERS 
  
 
Acute kidney injury 
 
  
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
  
 
Cough 
 
 Cough (Gr 2) 
 
Dyspnea 
 
 Dyspnea (Gr 2) 
 
Oropharyngeal pain 
 
  
 
 
Pneumonitis 
  
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
  
Pruritus 
 
 
 Pruritus (Gr 2)  
Rash maculo-papular 
 
 
 Rash maculo-papular (Gr 
2) 
 
1This table will be updated as the toxicity profile of the agent is revised.  Updates will be distributed to all 
Principal Investigators at the time of revision.  The current version can be obtained by contacting 
PIO@CTEP.NCI.NIH.GOV. Your name, the name of the investigator, the protocol and the agent should 
be included in the e-mail. 
 
Adverse events reported on MLN0128 (TAK-228) trials, but for which there is insufficient evidence 
to suggest that there was a reasonable possibility that MLN0128 (TAK-228) caused the adverse 
event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Blood and lymphatic system disorders - Other 

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57 
 
(hyperviscosity syndrome); Blood and lymphatic system disorders - Other (Raynaud's phenomenon); 
Febrile neutropenia 
CARDIAC DISORDERS - Heart failure; Pericardial effusion; Sinus tachycardia; Ventricular arrhythmia 
EYE DISORDERS - Blurred vision; Eye pain; Photophobia; Vision decreased 
GASTROINTESTINAL DISORDERS - Abdominal distension; Colitis; Dysphagia; Esophagitis; Gastritis; 
Gastroesophageal reflux disease; Gastrointestinal disorders - Other (intestinal obstruction); 
Gastrointestinal disorders - Other (intestinal perforation);  Gastrointestinal disorders - Other (salivary 
hypersecretion); Hemorrhoids; Ileus; Oral pain; Pancreatitis; Small intestinal obstruction; Small intestinal 
perforation; Toothache 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Chills; Flu like symptoms; Gait 
disturbance; General disorders and administration site conditions - Other (groin pain); Malaise; Non-
cardiac chest pain; Pain 
HEPATOBILIARY DISORDERS - Gallbladder obstruction 
IMMUNE SYSTEM DISORDERS - Allergic reaction 
INFECTIONS AND INFESTATIONS - Abdominal infection; Infections and infestations - Other (cystitis); 
Infections and infestations - Other (lower respiratory tract infection); Infections and infestations - Other 
(mucosal infection); Infections and infestations - Other (parotid gland); Kidney infection; Lung infection; 
Papulopustular rash; Sepsis; Skin infection; Upper respiratory infection 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS - Fall; Fracture; Injury, poisoning and 
procedural complications - Other (accidental overdose); Injury, poisoning and procedural complications - 
Other (postoperative fever); Injury, poisoning and procedural complications - Other (subdural 
hemorrhage); Tracheal obstruction 
INVESTIGATIONS - Alanine aminotransferase increased; Alkaline phosphatase increased; Aspartate 
aminotransferase increased; Blood bilirubin increased; Blood lactate dehydrogenase increased; 
Cholesterol high; GGT increased; Lipase increased; Lymphocyte count decreased; Neutrophil count 
decreased; White blood cell decreased 
METABOLISM AND NUTRITION DISORDERS - Acidosis; Hypercalcemia; Hyperkalemia; 
Hypernatremia; Hypertriglyceridemia; Hyperuricemia; Hypoalbuminemia; Hypocalcemia; Hyponatremia; 
Metabolism and nutrition disorders - Other (severe chronic malnutrition); Metabolism and nutrition 
disorders - Other (vitamin D deficiency)  
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Bone pain; Chest wall pain; Flank 
pain; Generalized muscle weakness; Muscle cramp; Myalgia 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) - Neoplasms 
benign, malignant and unspecified (incl cysts and polyps) - Other (non-hodgkin lymphoma); Treatment 
related secondary malignancy 
NERVOUS SYSTEM DISORDERS - Ataxia; Intracranial hemorrhage; Lethargy; Nervous system 
disorders - Other (carotid artery occlusion); Nervous system disorders - Other (neuropathy peripheral); 
Paresthesia; Radiculitis; Stroke; Tremor 
PSYCHIATRIC DISORDERS - Anxiety; Confusion; Depression; Psychiatric disorders - Other (mental 
status changes) 
RENAL AND URINARY DISORDERS - Dysuria; Hematuria; Proteinuria; Renal and urinary disorders - 
Other (strangury) 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Vaginal hemorrhage 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Bronchopulmonary hemorrhage; 
Epistaxis; Hiccups; Hypoxia; Nasal congestion; Pleural effusion; Pleuritic pain; Pneumothorax; Postnasal 
drip; Productive cough 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Dry skin; Hyperhidrosis; Rash 
acneiform; Urticaria 
VASCULAR DISORDERS - Flushing; Hypertension; Hypotension; Thromboembolic event 
 
Note: MLN0128 (TAK-228) in combination with other agents could cause an exacerbation of any adverse 
event currently known to be caused by the other agent, or the combination may result in events never 
previously associated with either agent. 
 
 
 

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58 
 
 
7.1.1.2 
CAEPR for Ziv-Aflibercept (VEGF-Trap, NSC # 724770) 
 
Version 2.8, April 17, 20191 
 
 Adverse Events with Possible  
 Relationship to Ziv-aflibercept (VEGF-Trap, AVE 0005) 
 (CTCAE 5.0 Term) 
[n= 941] 
 
 
 Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
  
 
Anemia 
 
 Anemia (Gr 2) 
 
 
Febrile neutropenia 
  
 
 
Hemolytic uremic 
syndrome 
  
 
 
Thrombotic 
thrombocytopenic 
purpura 
  
CARDIAC DISORDERS 
  
 
 
Cardiac disorders - Other 
(intracardiac thrombus) 
  
 
 
Chest pain - cardiac 
  
 
 
Myocardial infarction 
  
 
 
Restrictive 
cardiomyopathy 
  
GASTROINTESTINAL DISORDERS 
  
 
Abdominal pain 
 
 Abdominal pain (Gr 2) 
 
Constipation 
 
 Constipation (Gr 2) 
 
Diarrhea 
 
 Diarrhea (Gr 2) 
 
 
Gastrointestinal fistula2 
  
 
 
Gastrointestinal 
perforation3 
  
 
Mucositis oral 
 
  
Nausea 
 
 
 Nausea (Gr 2) 
 
Vomiting 
 
 Vomiting (Gr 2) 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
  
 
Edema limbs 
 
 Edema limbs (Gr 2) 
Fatigue 
 
 
 Fatigue (Gr 3) 
 
Fever 
 
 Fever (Gr 2) 
 
Pain 
 
  
INJURY, POISONING AND PROCEDURAL COMPLICATIONS 
  
 
Wound complication 
 
  
INVESTIGATIONS 
  
 
Alanine aminotransferase 
increased 
 
  
 
Alkaline phosphatase 
increased 
 
  
 
Aspartate 
aminotransferase 
increased 
 
  
 
Creatinine increased 
 
  

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59 
 
 
 Adverse Events with Possible  
 Relationship to Ziv-aflibercept (VEGF-Trap, AVE 0005) 
 (CTCAE 5.0 Term) 
[n= 941] 
 
 
 Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
 
Lymphocyte count 
decreased 
 
 Lymphocyte count decreased 
(Gr 4) 
 
Neutrophil count 
decreased 
 
 Neutrophil count decreased (Gr 
4) 
 
Platelet count decreased  
 Platelet count decreased (Gr 4) 
 
Weight loss 
 
  
 
White blood cell 
decreased 
 
 White blood cell decreased (Gr 
2) 
METABOLISM AND NUTRITION DISORDERS 
  
 
Anorexia 
 
 Anorexia (Gr 2) 
 
Dehydration 
 
  
 
Hyponatremia 
 
  
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
  
 
Arthralgia 
 
 Arthralgia (Gr 2) 
 
Myalgia 
 
 Myalgia (Gr 2) 
NERVOUS SYSTEM DISORDERS 
  
Headache 
 
 
 Headache (Gr 3) 
 
 
Ischemia cerebrovascular  Ischemia cerebrovascular (Gr 2) 
 
 
Reversible posterior 
leukoencephalopathy 
syndrome 
  
 
 
Transient ischemic attacks   
RENAL AND URINARY DISORDERS 
  
 
 
Acute kidney injury 
  
 
 
Nephrotic syndrome 
  
Proteinuria 
 
 
 Proteinuria (Gr 3) 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS 
  
 
Genitourinary system 
fistula4 
 
  
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
  
 
Cough 
 
 Cough (Gr 2) 
 
Dyspnea 
 
 Dyspnea (Gr 3) 
 
Rhinorrhea 
 
  
Voice alteration 
 
 
 Voice alteration (Gr 2) 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
  
 
Alopecia 
 
  
 
Palmar-plantar 
erythrodysesthesia 
syndrome 
 
  
 
Skin hyperpigmentation 
 
  
VASCULAR DISORDERS 
  
 
 
Arterial 
thromboembolism 
  
Hypertension 
 
 
 Hypertension (Gr 3) 
 
Thromboembolic event 
 
 Thromboembolic event (Gr 3) 

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60 
 
 
 Adverse Events with Possible  
 Relationship to Ziv-aflibercept (VEGF-Trap, AVE 0005) 
 (CTCAE 5.0 Term) 
[n= 941] 
 
 
 Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)  
 
Vascular disorders - Other 
(hemorrhage)5 
 
 Vascular disorders - Other 
(hemorrhage) (Gr 4) 
 
1This table will be updated as the toxicity profile of the agent is revised. Updates will be 
distributed to all Principal Investigators at the time of revision. The current version can be 
obtained by contacting  PIO@CTEP.NCI.NIG.GOV.  Your name, the name of the investigator, 
the protocol and the agent should be included in the e-mail. 
 
2Gastrointestinal fistulas may include: Anal fistula, Colonic fistula, Duodenal fistula, Esophageal 
fistula, Gastric fistula, Gastrointestinal fistula, Rectal fistula, and other sites under the 
GASTROINTESTINAL DISORDERS SOC. 
 
3Gastrointestinal perforation may include: Colonic perforation, Duodenal perforation, 
Esophageal perforation, Gastric perforation, Ileal perforation, Jejunal perforation, Rectal 
perforation, and Small intestinal perforation under the GASTROINTESTINAL DISORDERS 
SOC. 
 
4Genitourinary fistulas may include: female genital tract fistula, uterine fistula, and vaginal 
fistula. 
 
5The majority of hemorrhage events were mild. Major events, defined as symptomatic bleeding 
in a critical area or organ (e.g., eye, GI hemorrhage, GU hemorrhage, respiratory hemorrhage), 
and nervous system [including fatal intracranial hemorrhage and cerebrovascular accident] have 
been reported. 
 
6Infection may include any of the 75 infection sites under the INFECTIONS AND 
INFESTATIONS SOC. 
 
Adverse events reported on ziv-aflibercept (VEGF-Trap, AVE 0005) trials, but for which 
there is insufficient evidence to suggest that there was a reasonable possibility that ziv-
aflibercept (VEGF-Trap, AVE 0005) caused the adverse event:  
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Hemolysis 
CARDIAC DISORDERS - Cardiac disorders - Other (left ventricular diastolic dysfunction); 
Heart failure; Left ventricular systolic dysfunction; Pericarditis; Supraventricular tachycardia 
EAR AND LABYRINTH DISORDERS - Tinnitus; Vertigo 
ENDOCRINE DISORDERS - Hyperthyroidism; Hypothyroidism 
EYE DISORDERS - Blurred vision; Extraocular muscle paresis; Eye disorders - Other 
(blindness transient); Eye disorders - Other (diplopia); Vitreous hemorrhage 
GASTROINTESTINAL DISORDERS - Abdominal distension; Anal mucositis; Belching; 
Colitis; Dental caries; Dry mouth; Dyspepsia; Dysphagia; Esophageal pain; Flatulence; Gastritis; 
Gastrointestinal disorders - Other (early satiety); Gastrointestinal disorders - Other 

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61 
 
(gastrointestinal necrosis); Gastrointestinal disorders - Other (hiatal hernia); Gastrointestinal 
disorders - Other (intestinal ischemia); Gastrointestinal disorders - Other (peritonitis); 
Gastrointestinal disorders - Other (pneumatosis intestinalis); Gingival pain; Hemorrhoids; Ileus; 
Oral pain; Rectal mucositis; Rectal ulcer; Small intestinal mucositis; Small intestinal obstruction 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Edema face; 
Edema trunk; Facial pain; Injection site reaction; Non-cardiac chest pain 
HEPATOBILIARY DISORDERS - Cholecystitis 
IMMUNE SYSTEM DISORDERS - Allergic reaction 
INFECTIONS AND INFESTATIONS - Infection6 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS - Biliary anastomotic 
leak; Gastric anastomotic leak; Infusion related reaction; Vascular access complication; Wound 
dehiscence 
INVESTIGATIONS - Activated partial thromboplastin time prolonged; Blood bilirubin 
increased; Blood lactate dehydrogenase increased; Cardiac troponin I increased; Ejection fraction 
decreased; GGT increased; Hemoglobin increased; Lipase increased; Serum amylase increased 
METABOLISM AND NUTRITION DISORDERS - Hypercalcemia; Hyperglycemia; 
Hyperkalemia; Hypoalbuminemia; Hypocalcemia; Hypoglycemia; Hypokalemia; 
Hypomagnesemia; Hypophosphatemia 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Arthritis; Avascular 
necrosis; Back pain; Chest wall pain; Generalized muscle weakness; Head soft tissue necrosis; 
Joint range of motion decreased; Muscle cramp; Muscle weakness upper limb; Musculoskeletal 
and connective tissue disorder - Other (musculoskeletal stiffness); Myositis; Neck pain; 
Osteonecrosis; Osteonecrosis of jaw; Pain in extremity; Pelvic soft tissue necrosis; Rotator cuff 
injury 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND 
POLYPS) - Tumor hemorrhage 
NERVOUS SYSTEM DISORDERS - Amnesia; Ataxia; Cognitive disturbance; Dizziness; 
Dysgeusia; Encephalopathy; Extrapyramidal disorder; Leukoencephalopathy; Memory 
impairment; Paresthesia; Peripheral sensory neuropathy; Seizure; Syncope; Vagus nerve disorder 
PSYCHIATRIC DISORDERS - Anxiety; Confusion; Depression; Insomnia; Psychiatric 
disorders - Other (mental status change); Psychosis 
RENAL AND URINARY DISORDERS - Hematuria 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Irregular menstruation 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Hoarseness; Hypoxia; 
Laryngeal mucositis; Nasal congestion; Pharyngeal mucositis; Pharyngolaryngeal pain; Pleural 
effusion; Pneumonitis; Pneumothorax; Pulmonary fibrosis; Respiratory, thoracic and mediastinal 
disorders - Other (nasal dryness); Respiratory, thoracic and mediastinal disorders - Other (septal 
perforation); Respiratory, thoracic and mediastinal disorders - Other (throat swelling); Tracheal 
fistula; Tracheal mucositis 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Dry skin; Hyperhidrosis; Nail loss; 
Rash acneiform; Rash maculo-papular; Skin and subcutaneous tissue disorders - Other 
(hyperemia); Skin ulceration 
VASCULAR DISORDERS - Hematoma; Hypotension; Peripheral ischemia; Phlebitis 
 
Note: Ziv-aflibercept (VEGF-Trap, AVE 0005) in combination with other agents could cause an 
exacerbation of any adverse event currently known to be caused by the other agent, or the 

NCI Protocol #: 9585 
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62 
 
combination may result in events never previously associated with either agent. 
 
 
 
 
7.2 
Adverse Event Characteristics 
 
• CTCAE term (AE description) and grade: The descriptions and grading scales 
found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) 
version 4.0 will be utilized until March 31, 2018 for AE reporting.  CTCAE version 5.0 
will be utilized for AE reporting beginning April 1, 2018. All appropriate treatment 
areas should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE 
version 5.0 can be downloaded from the CTEP web site  
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
• 
For expedited reporting purposes only: 
- 
AEs for the agent that are bold and italicized in the CAEPR (i.e., those listed in the 
SPEER column, Section 7.1.1) should be reported through CTEP-AERS only if the 
grade is above the grade provided in the SPEER. 
- 
Other AEs for the protocol that do not require expedited reporting are outlined 
in section 7.3.4. 
 
• Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 
Expedited Adverse Event Reporting 
 
7.3.1 Expedited AE reporting for this study must use CTEP-AERS (CTEP Adverse Event 
Reporting System), accessed via the CTEP Web site (http://ctep.cancer.gov). The reporting 
procedures to be followed are presented in the “NCI Guidelines for Investigators: Adverse 
Event Reporting Requirements for DCTD (CTEP and CIP) and DCP INDs and IDEs” which can 
be downloaded from the CTEP Web site (http://ctep.cancer.gov).  These requirements are briefly 
outlined in the tables below (Section 7.3.3). 
 
In the rare occurrence when Internet connectivity is lost, a 24-hour notification is to be made to 
CTEP by telephone at 301-897-7497. Once Internet connectivity is restored, the 24-hour 
notification phoned in must be entered electronically into CTEP-AERS by the original submitter 
at the site. 
 
7.3.2 CTEP-AERS is programmed for automatic electronic distribution of reports to the 
following individuals:  Study Coordinator of the Lead Organization, Principal Investigator, and 
the local treating physician. CTEP-AERS provides a copy feature for other e-mail recipients. 
 

NCI Protocol #: 9585 
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63 
 
7.3.3 Expedited Reporting Guidelines 
 
Use the NCI protocol number and the protocol-specific patient ID assigned during trial 
registration on all reports. 
 
Note: A death on study requires both routine and expedited reporting regardless of 
causality, unless as noted below.  Attribution to treatment or other cause must be 
provided. 
 
Death due to progressive disease should be reported as Grade 5 “Disease progression” in the 
system organ class (SOC) “General disorders and administration site conditions.” Evidence that 
the death was a manifestation of underlying disease (e.g., radiological changes suggesting tumor 
growth or progression: clinical deterioration associated with a disease process) should be 
submitted. 
 
Phase 1 and Early Phase 2 Studies: Expedited Reporting Requirements for Adverse 
Events that Occur on Studies under an IND/IDE within 30 Days of the Last Administration 
of the Investigational Agent/Intervention 1, 2 
 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE: Investigators MUST immediately report to the sponsor (NCI) ANY Serious Adverse Events, whether or not 
they are considered related to the investigational agent(s)/intervention (21 CFR 312.64) 
An adverse event is considered serious if it results in ANY of the following outcomes: 
1) 
Death 
2) 
A life-threatening adverse event 
3) 
An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 
hours 
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions 
5) 
A congenital anomaly/birth defect. 
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon medical judgment, they may jeopardize the patient or 
subject and may require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the NCI via CTEP- 
AERS within the timeframes detailed in the table below. 
Hospitalization 
Grade 1 and Grade 2 Timeframes 
Grade 3-5 
Timeframes 
Resulting in 
Hospitalization 
≥ 24 hrs 
10 Calendar Days 
24-Hour 5 Calendar 
Days
Not resulting in 
Hospitalization 
≥ 24 hrs 
Not required 

NCI Protocol #: 9585 
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64 
 
 
 
7.4 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions. AEs reported 
through CTEP-AERS must also be reported in routine study data submissions. 
 
7.5 
Secondary Malignancy 
 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (e.g., 
treatment with investigational agent/intervention, radiation or chemotherapy). A secondary 
malignancy is not considered a metastasis of the initial neoplasm. 
CTEP requires all secondary malignancies that occur following treatment with an agent under 
an NCI IND/IDE be reported via CTEP-AERS. Three options are available to describe the 
event: 
 
• Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic 
leukemia [AML]) 
• Myelodysplastic syndrome (MDS) 
• Treatment-related secondary malignancy 
 
Any malignancy possibly related to cancer treatment (including AML/MDS) should also be 
reported via the routine reporting mechanisms outlined in each protocol. 
 
7.6 
Second Malignancy 
 
A second malignancy is one unrelated to the treatment of a prior malignancy (and is NOT a 
metastasis from the initial malignancy).  Second malignancies require ONLY routine 
reporting via CDUS unless otherwise specified. 
NOTE: Protocol specific exceptions to expedited reporting of serious adverse events are found in the Specific 
Protocol Exceptions to Expedited Reporting (SPEER) portion of the CAEPR. 
Expedited AE reporting timelines are defined as: 
o
“24-Hour; 5 Calendar Days” - The AE must initially be reported via CTEP-AERS within 24 hours of learning 
of the AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour report. 
o
“10 Calendar Days” - A complete expedited report on the AE must be submitted within 10 calendar days of 
learning of the AE. 
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention and have an attribution of possible, probable, or definite require reporting as follows: 
Expedited 24-hour notification followed by complete report within 5 calendar days for: 
•
All Grade 3, 4, and Grade 5 AEs 
Expedited 10 calendar day reports for: 
•
Grade 2 AEs resulting in hospitalization or prolongation of hospitalization 
2For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-lives, 
rounded UP to the nearest whole day, after the agent/intervention was last administered. Footnote “1” above 
applies after this reporting period. 
Effective Date: May 5, 2011 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
65 
 
 
8. PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational agents 
administered in this study can be found in Section 7.1. 
 
8.1 
CTEP IND Agent(s) 
 
8.1.1 MLN0128 (TAK-228) (NSC #768435) 
 
Other Names: TAK-228, INK128 
Chemical Name: 3-(2-amino-1,3-benzoxazol-5-yl)-1-(propan-2-yl)-1Hpyrazolo [3, 4-d] 
pyrimidin-4-amine 
 
Classification: mTOR inhibitor, TORC1/2 
 
CAS Registry Number: 1224844 
 
Molecular Formula: C15H15N7O 
M.W.: 309.3 
 
Approximate Solubility: MLN0128 (TAK-228) exhibits a pH-dependent aqueous solubility: 
At physiological pH, the solubility is approximately 0.1 mg/mL; 
At or below pH 3, the solubility is greater than 15 mg/mL. 
 
Mode of Action: MLN0128 (TAK-228) is a non-rapamycin analog mTOR (mechanistic target of 
rapamycin) kinase inhibitor.  The mTOR kinase regulates cell growth, translational control, 
angiogenesis, and cell survival by integrating nutrient and hormonal signals.  The mTOR 
complex (TORC) is an intracellular point of convergence for a number of cellular signaling 
pathways.  ML0128 is a potent and selective adenosine tri-phosphate (ATP)-competitive inhibitor 
of mTOR complex 1 and 2 (TORC1/2). 
 
Description: White to off-white, crystalline powder. 
 
How Supplied: MLN0128 (TAK-228) is supplied by Millennium Pharmaceuticals, Inc. and 
distributed by the Pharmaceutical Management Branch, CTEP/DCTD/NCI as size 2 hard gelatin 
capsules in the following strengths: 1 mg (white opaque color) and 3 mg (orange opaque color) 
The 5 mg (gray opaque color) will not be utilized in the protocol. The composition of the drug 
product consists of a blend of MLN0128 drug substance, microcrystalline cellulose, and 
magnesium stearate.  Milled formulations will have a white label with a large watermark of the 
strength on the label. As of Amendment 13/Version 07, all newly enrolled patients will be given 
the milled formulation of MLN0128 (TAK-228).  
 
MLN0128 capsules are packaged in 30-count, 60-cc high-density polyethylene (HDPE), white, 
opaque, round, tamper- and child-resistant bottles.   
 
Storage: Capsules are to be stored in the original package between 15°C to 30°C, with allowed 
short-term excursions between 2°C and 40°C. 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
66 
 
 
Route of Administration: Orally, given 1 hour before or 2 hours after a meal.  Do not chew, open 
or manipulate the capsule in any way prior to swallowing.  Each dose should be taken with 8 ounces 
(240 ml) of water. 
 
Potential Drug Interactions: Multiple human metabolizing enzymes are involved in 
themetabolism of MLN0128 (TAK-228).  Recently completed in vitro metabolism experiments 
in human hepatocytes using 14C-labeled TAK-228 suggest that TAK-228 is metabolized 
primarily via CYP1A2 (approximately 31%-40%), with a minor contribution from CYP3A4 
(approximately 11%-22%). These data suggest that TAK-228 is also metabolized by direct 
glucuronidation (approximately 22%) and an unidentified non-uridine diphosphate glucuronosyl 
transferase pathway (approximately 18%). The new data differ from the previous in vitro CYP 
phenotyping data obtained using recombinant CYP enzymes, which suggested the involvement 
of CYP2C9 (approximately 35%), CYP2C19 (approximately 28%), and CYP3A4 
(approximately 28%) in TAK-228 metabolism. In addition, physiologically based PK modeling 
and simulation using the new metabolism data for TAK-228 suggest that the risk for a 
metabolism-based drug-drug interaction with TAK-228 appears to be low. 
 
Patient Care Implications: 
Women of childbearing potential should use effective methods of contraception during and through 
90 days after the last dose of MLN0128. 
 
Men should use effective methods of contraception and not donate sperm during and through 120 
days after the last dose of MLN0128. 
 
Availability 
MLN0128 (TAK-228) is provided to the NCI under a Collaborative Agreement between the 
Pharmaceutical Collaborator and the DCTD, NCI (see Section 12.3). 
 
NCI-supplied agents may be requested by the Principal Investigator (or their authorized 
designee) at each participating institution.  Pharmaceutical Management Branch (PMB) policy 
requires that agent be shipped directly to the institution where the patient is to be treated.  PMB 
does not permit the transfer of agents between institutions (unless prior approval from PMB is 
obtained).  The CTEP-assigned protocol number must be used for ordering all CTEP-supplied 
investigational agents.  The responsible investigator at each participating institution must be 
registered with CTEP, DCTD through an annual submission of FDA Form 1572 (Statement of 
Investigator), Curriculum Vitae, Supplemental Investigator Data Form (IDF), and Financial 
Disclosure Form (FDF).  If there are several participating investigators at one institution, CTEP-
supplied investigational agents for the study should be ordered under the name of one lead 
investigator at that institution. 
 
In general, sites may order initial agent supplies when a subject is being screened for enrollment 
onto the study. 
 
Active CTEP-registered investigators and investigator-designated shipping designees and 
ordering designees can submit agent requests through the PMB Online Agent Order Processing 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
67 
 
(OAOP) application.  Access to OAOP requires the establishment of a CTEP Identity and Access 
Management (IAM) account and the maintenance of an “active” account status and a “current” 
password.  For questions about drug orders, transfers, returns, or accountability, call or email 
PMB any time.  Refer to the PMB’s website for specific policies and guidelines related to agent 
management.   
 
Agent Inventory Records – The investigator, or a responsible party designated by the 
investigator, must maintain a careful record of the receipt, dispensing and final disposition of all 
agents received from the PMB using the appropriate NCI Investigational Agent (Drug) 
Accountability Record (DARF) available on the CTEP forms page.  Store and maintain separate 
NCI Investigational Agent Accountability Records for each agent, strength, formulation and 
ordering investigator on this protocol. 
 
Useful Links and Contacts 
• 
CTEP Forms, Templates, Documents:  http://ctep.cancer.gov/forms/ 
• 
NCI CTEP Investigator Registration:  PMBRegPend@ctep.nci.nih.gov 
• 
PMB policies and guidelines: 
http://ctep.cancer.gov/branches/pmb/agent_management.htm  
• 
PMB Online Agent Order Processing (OAOP) application: https://eapps-
ctep.nci.nih.gov/OAOP/pages/login.jspx  
• 
CTEP Identity and Access Management (IAM) account: https://eapps-
ctep.nci.nih.gov/iam/ 
• 
CTEP Associate Registration and IAM account help:  ctepreghelp@ctep.nci.nih.gov  
• 
PMB email:  PMBAfterHours@mail.nih.gov 
• 
PMB phone and hours of service: (240) 276-6575 Monday through Friday between 8:30 
am and 4:30 pm (ET). 
 
 
8.1.2 Ziv-Aflibercept (NSC #724770) 
 
Other Names: aflibercept, AVE0005, ziv-aflibercept 
 
Classification: Recombinant humanized fusion protein (Chinese hamster ovary source). 
 
M.W.: 115 kDa 
 
Approximate Solubility: at least 100mg/ml water (with 5 mM Na phosphate, 5 mM Na 
citrate, 100 mM NaCl, 0.004-0.08% Polysorbate 20) at 5°C and 25°C. 
 
Mode of Action: The cytokine VEGF binds to and activates VEGFR1 and VEGFR2 on the 
vascular endothelium, promoting new vessel formation. VEGF Trap is a soluble 
recombinant decoy receptor that binds and inactivates extravascular and hematologic VEGF. It 
reduces tumor vasculature density, available nutrient supply, and tissue matrix components 
escaping from leaky tumor vessels. 
 
Description: The fusion protein VEGF Trap is 2 portions of human VEGF receptors 

NCI Protocol #: 9585 
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68 
 
extracellular domains, VEGFR1 and VEGFR2, fused to the Fc portion of human IgG1. 
 
How Supplied: VEGF Trap is supplied by Sanofi-Aventis Pharmaceuticals and distributed by 
the CTEP, DCTD, NCI.  VEGF Trap is a sterile, nonpyrogenic, colorless to pale yellow solution 
in vials of 100 mg (4 mL) or 200 mg (8 mL) at a concentration of 25 mg/mL. The solution 
contains the following excipients: sucrose, sodium chloride, sodium citrate dihydrate, citric acid 
monohydrate, polysorbate 20, sodium phosphate dibasic heptahydrate, sodium phosphate 
monobasic monohydrate, and water for injection. The pH of VEGF Trap is 6.2. The product is 
packaged in a type 1, clear borosilicate glass vial closed with a flanged cap with tear-off lid and 
inserted sealing disc, Flurotec® (PTFE) coated. 
 
Preparation: Prior to infusion, the VEGF Trap dosage form must be diluted directly into 
infusion bags of 0.9% sodium chloride solution or 5% dextrose. The concentration of the diluted 
solution can range between 0.6 and 8 mg/mL. The pH of the diluted solution is about 6.2. 
 
Storage: Store intact vials in the refrigerator (2o to 8o C). 
 
Stability: Shelf life stability studies of intact vials are ongoing. VEGF Trap’s provisional shelf-life 
is up to 36 months at 2o to 8°C. 
 
Caution: The sterile single use vials contain no antibacterial preservatives. Discard remaining 
agent 8 hours after initial entry. 
 
VEGF Trap diluted to a concentration of 0.6 to 8 mg/mL in 0.9% NaCl or 5% dextrose has 
demonstrated chemical and physical stability for up to 24 hours under refrigerated conditions (2 to 
8°C) or up to 8 hours at ambient temperature (approximately 25°C) in infusion bags made of 
polyvinyl chloride (PVC) containing di(2-ethylhexyl)phthalate (DEHP) or polyolefin (PVC free 
DEHP free). 
 
Route of Administration: Intravenous 
 
Method of Administration: : Administer VEGF Trap intravenously over 1 hour into a peripheral 
vein or central venous catheter using gravity, an infusion pump, or syringe pump. The infusion 
should not exceed two hours at room temperature (approximately 25°C). 
 
VEGF Trap may be administered using infusion tubing made of PVC containing DEHP, 
polyethylene lined PVC, DEHP free PVC containing tris (2-ethylhexyl) trimellitate (TOTM), 
polypropylene, or polyurethane. 
 
The infusion set must contain a 0.2 micron polyethersulfone inline filter.  Polyvinylidene fluoride 
(PVDF) filters or Nylon filters should not be used. 
 
Availability 
 
Ziv-Aflibercept is an investigational agent supplied to investigators by the Division of Cancer 
Treatment and Diagnosis (DCTD), NCI. 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
69 
 
 
Ziv-Aflibercept is provided to the NCI under a Collaborative Agreement between the 
Pharmaceutical Collaborator and the DCTD, NCI (see Section 12.3). 
 
8.1.3 Agent Ordering and Agent Accountability 
 
8.1.3.1 NCI-supplied agents may be requested by the Principal Investigator (or their authorized 
designee) at each participating institution.  Pharmaceutical Management Branch (PMB) policy 
requires that agent be shipped directly to the institution where the patient is to be treated. PMB 
does not permit the transfer of agents between institutions (unless prior approval from PMB is 
obtained). The CTEP-assigned protocol number must be used for ordering all CTEP-supplied 
investigational agents. The responsible investigator at each participating institution must be 
registered with CTEP, DCTD through an annual submission of FDA Form 1572 (Statement of 
Investigator), Curriculum Vitae, Supplemental Investigator Data Form (IDF), and Financial 
Disclosure Form (FDF).  If there are several participating investigators at one institution, CTEP-
supplied investigational agents for the study should be ordered under the name of one lead 
investigator at that institution. 
 
Active CTEP-registered investigators and investigator-designated shipping designees and 
ordering designees can submit agent requests through the PMB Online Agent Order Processing 
(OAOP) application (https://eapps-  ctep.nci.nih.gov/OAOP/pages/login.jspx). Access to OAOP 
requires the establishment of a CTEP Identity and Access Management (IAM) account 
(https://eapps-ctep.nci.nih.gov/iam/) and the maintenance of an “active” account status and a 
“current” password.  For questions about drug orders, transfers, returns, or accountability, call 
(240) 276-6575 Monday through Friday between 8:30 am and 4:30 pm (ET) or email 
PMBAfterHours@mail.nih.gov anytime. 
 
8.1.3.2 Agent Inventory Records – The investigator, or a responsible party designated by the 
investigator, must maintain a careful record of the inventory and disposition of all agents 
received from DCTD using the NCI Drug Accountability Record Form (DARF).  (See the NCI 
Investigator’s Handbook for Procedures for Drug Accountability and Storage.) 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
 
Biologic activity of ziv-aflibercept may be difficult to assess because it may often seem primarily 
cytostatic rather than cytotoxic in function. When anti-VEGF agents are used in a 
monotherapeutic fashion, only a small number of patients have a major tumor response (Beaudry  
et al., 2005; Davis et al., 2003). To advance the clinical testing of ziv-aflibercept and the 
projected additional benefits of combination with MLN0128 (TAK-228), biopsies will be 
performed. These studies are designed to determine if combining ziv-aflibercept and MLN0128 
(TAK-228) will inhibit both the phosphorylation of effectors that are downstream of the targets 
of the drugs as well as potentiating anti-angiogenic activities. 
 
For the expansion cohorts, we will be performing correlative studies in 10 patients. There will be 
discussion between the PI and NCI-CTEP to further expand with additional patients. Our 
interventional radiologists will coordinate with our study radiologist. Our study radiologist will 
identify an optimal area for interventional radiologists to biopsy. Biopsies will be performed at 

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70 
 
baseline, on Cycle 1 Day 8, and at time of progression. All the treating investigators will be 
educated and trained to report to the principal investigator the first signs of progression. For the 
Biomarker Assay Characterization for RPPA and IHC, please see Appendix F. 
 
 
 
9.1 
Immunohistochemistry (IHC) 
 
Dr. Funda Meric-Bernstam’s (Chairman of the Department of Investigational Cancer 
Therapeutics at M. D. Anderson) laboratory will evaluate protein expression (both the total and 
phosphorylated proteins) by immunohistochemical (IHC) staining on slides from fresh tumor 
samples as well as archival tissue material (if available).  Multiple predictive biomarkers 
relevant to the mechanism of action of mTOR inhibitors and VEGF-trap inhibitors will be 
evaluated in the tumor specimens. 
 
Expression/phosphorylation of potential markers of response (including but not limited to AKT, 
pAKTSer473, pS6 and PTEN) and markers of angiogenesis (including CD31) will be detected 
in the tumors using immunohistochemistry and correlated with clinical tumor response.  IHC 
will also be used to access microvessel density.  Archival tissue and pre- treatment/post-
treatment biopsies will be evaluated by H&E. Treatment-induced apoptosis will be assessed 
with IHC for cleaved caspase (Meric-Bernstam et al., 2004). 
The reason we chose IHC as a tool to evaluate response to new drug before and after therapy is 
because most biological processes and functions occur at the protein level. Ultimately, protein 
functional status and its expression level in cancer may provide the best assessment for 
therapeutic development. 
 
The current standard method in diagnostic pathology is a conventional semiquantitative visual 
analysis of brown stain immunohistochemical staining (DAB based) with a light microscope. 
 
In the event that tissue material is limited, we will give priority to RPPA analysis as RPPA is an 
extremely effective and sensitive method for measuring multiple proteins in cancer cells and 
this technique can be applied to tiny amounts of material. Dr. Funda Meric-Bernstam and Dr. 
Gordon Mills’ laboratory at MD Anderson Cancer Center has established this method, and we 
are confident that we can use this technology to measure all the markers of relevance to this 
study. However, when it is possible to perform both RPPA and IHC for the same proteins, the 
results from each platform will validate each other. 
 
9.1.1 Outcome measure 
 
Total and phosphorylated expression measurements including (but not limited to): 
pAKTSer473, pS6, PTEN, CD31. 
 
9.1.2 Assessment 
 
IHC measurement will be based on percent of positive staining cells under microscope and 
intensity of positive cells.  Intensity will be ranked as 0: no staining, 1: weak staining, 2: 
moderate staining 3: strong staining. The intensity is multiplied by the percentage of positive 

NCI Protocol #: 9585 
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71 
 
cells. This is called H-score, which will correlate to therapeutic response. 
 
Timing of Assessment 
 
Samples will be collected at baseline, Day 8 of Cycle 1, and at progression. 
 
After all samples have been collected, tissue samples will be processed no later than 3 months 
from the time of collection at Dr. Funda Meric-Bernstam’s laboratory.  Basic data analysis will 
be performed no later than 2 months after the time of slide staining. 
 
9.1.3 Data recording 
 
The slide will be read by the following day after IHC and imaging data will be transferred to 
computer after read. 
 
9.1.4 Collection of specimens 
 
Please refer to Appendix G. 
 
9.1.5 Site performing correlative study 
 
The University of Texas MD Anderson Cancer Center in Houston, Texas, in the Division of 
Cancer Medicine (Departments of Investigational Cancer Therapeutics and Systems Biology) 
will be performing the IHC. 
 
9.2 
Reverse Phase Protein Assay (RPPA) 
 
Dr. Funda Meric-Bernstam’s (Chairman of the Department of Investigational Cancer 
Therapeutics at M. D. Anderson) laboratory will perform Reverse Phase Protein Arrays 
(RPPAs) on peripheral blood mononuclear cells (PBMCs), platelet-enriched plasma samples, 
and tumor biopsies to quantitate protein expression/phosphorylation, as well as proliferation, 
cell cycle progression, and apoptosis markers.  Our priority will be looking at molecular 
alterations in the mTOR signaling pathway (including but not limited to AKT, pAKTSer473, 
pS6, and PTEN) and markers of angiogenesis. 
 
A number of blood vessel-specific markers will be quantified using RPPA analysis. CD31 is a 
particularly useful marker for quantifying angiogenesis by RPPA. Decreased CD31 levels can 
provide a surrogate for inhibition of angiogenesis. Other blood vessel-specific markers that can 
serve as surrogates for angiogenesis and can be quantified using RPPA include VEGF receptors 
VEGFR1 and VEGFR2. We have identified and validated antibodies that detect CD31, 
VEGFR1 and VEGFR2 for RPPA. The arrayers, robotics and robust algorithms are available to 
efficiently utilize the technologies. 
 
Assessment of MLN0128 (TAK-228) alone, ziv-aflibercept alone and MLN0128 (TAK-228) + 
ziv-aflibercept combination administration will be assayed by RPPA on patient PBMCs and 
tumor biopsies.  Specimens will be analyzed for (1) effect on target proteins or proteins 

NCI Protocol #: 9585 
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72 
 
associated with the target protein upon administration of each drug alone or combination of 
drugs (2) effect on biological markers (e.g., proliferation, apoptosis, etc.) (3) potential off-target 
effects (4) association of protein levels (basal or treated) or post-translational modifications 
with patient outcome. 
 
 
9.2.1 Outcome Measure 
 
Total and phosphorylated expression measurements including (but not limited to): 
pAKTSer473, pS6, PTEN, CD31. 
 
9.2.2 Assessment 
 
Method of Assessment 
Flash-frozen tumor tissues (obtained as outlined in Appendix G) will be quickly cut (if 
necessary) and weighed; tumor samples with a mass range of 10-40 mg are placed in ice-cold 
lysis buffer (1% Triton X100, 50mM Hepes, pH 7.4, 150mM NaCl, 1.5mM MgCl2, 1mM 
EGTA, 100mM NaF, 10mM NaPPi, 10% glycerol, freshly added Roche Complete tablet, freshly 
added 1mM Na3VO4) at 1mL/40mg tissue, and the sample will be homogenized on ice. PBMC 
pellets (obtained as outlined in Appendix H) will be lysed in a similar fashion.  Protein 
estimation of supernatants collected from lysed tissue will be determined by bicinchroninic acid 
assay. Proteins in sample will be denatured and linearized upon addition of SDS sample buffer 
(35% Glycerol, 8% SDS, 0.25M Tris-HCL, pH 6.8, 10% 2-mercaptoethanol) and boiling. 
 
Sample concentrations will be adjusted to ~1mg/ml using dilution buffer (3 parts lysis buffer: 1 
part SDS buffer). Samples will be serially diluted 2-fold at least 5 times, and spotted onto 
nitrocellulose-coated microscope (FAST) slides. Relative levels of proteins or protein 
modifications will be detected by amplifying signal with streptavidin-biotin and tyramide and 
colorimetric development by diaminobenzidine. Stained slides will be scanned for quantitation 
by a 24+-bit, CCD flatbed scanner at 600+ dpi in grayscale and saved as a 16-bit tiff image. 
Relative levels of total or post- translationally modified proteins will be quantitated by software 
developed for RPPA (MicroVigene and supercurve script written for R). Other analyses will be 
performed as appropriate, including supervised and unsupervised hierarchical clustering of 
samples and proteins. 
 
Timing of Assessment 
Samples will be collected at baseline, Day 8 of Cycle 1, and at progression. 
 
After all samples have been collected, tissue samples will be processed no later than 3 months 
from the time of collection at Dr. Funda Meric-Bernstam’s laboratory.  RPPA sample dilution, 
slide printing and staining will be performed no later than 3 months after the time of tissue 
processing. Basic data analysis will be performed no later than 2 months after the time of slide 
staining. 
 
9.2.3 Data Recording 
 

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73 
 
Method of Recording 
All files created (xls, tif, txt, ppt) are currently stored (password protected) in the Systems 
Biology server, which is backed up in designated space within the institution. 
 
Timing of Recording 
Data will be recorded in the server no later than one week after data has been acquired. 
 
9.2.4 Collection of Specimen(s) 
 
Please refer to Appendix G and Appendix H. 
 
9.2.5 Handling of Specimen(s) 
 
Please refer to Appendix G and Appendix H. 
 
9.2.6 Shipping of Specimen(s) 
 
The specimens will not be shipped to another location. All correlative studies will be performed 
at The University of Texas MD Anderson Cancer Center in Houston, Texas, in the Division of 
Cancer Medicine (Departments of Investigational Cancer Therapeutics and Systems Biology). 
 
9.2.7 Site(s) Performing Correlative Study 
 
The University of Texas MD Anderson Cancer Center in Houston, Texas, in the Division of 
Cancer Medicine (Departments of Investigational Cancer Therapeutics and Systems Biology) 
will be performing the IHC. 
 
 

NCI Protocol #: 9585 
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74 
 
 
10. STUDY CALENDAR 
Baseline evaluations are to be conducted within 1 week prior to start of protocol therapy. Scans and x-rays must be done <4 weeks 
(28 days) prior to the start of therapy.  In the event that the patient’s condition is deteriorating, laboratory evaluations should be 
repeated within 48 hours prior to initiation of the next cycle of therapy.  Routine lab studies, vital signs, weight, height, and 
performance status must be done within 14 days of Day 1 and will have a flexibility window of +/- 2 days. Any re-staging/disease 
assessment criteria will have a flexibility window of +/- 3 days. Dosing can be given up to 2 days after physical exam as long as it 
has not been less than 7 days from last dose. However, this will not be routine practice and will only occur in special circumstances 
as deemed appropriate by PI. There will be a +/-2 day flexibility window for assessments on Day 1, Day 8, Day 15, and Day 22 of 
each cycle. Both study drugs will have a +/- 2 day window. If the ziv-aflibercept dosing is rescheduled, then the MLN0128 
(TAK-228) dosing will be adjusted accordingly. 
Screening 
Cycle 1 
Cycle 2 
Cycle 3 and Beyond 
Off Studyf 
Within 
28 
Days 
Within 
14 
Days 
Within 
7 
Days 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
MLN0128  
(TAK-228) 
A------------ 3 Days On and 4 Days Off Oral Starting Day 2 of Every Cycle----------------A 
Ziv-Aflibercept 
B 
B 
B 
B 
B 
B 
Informed 
consent 
X 
Demographics 
X 
Medical history 
X 
Concurrent 
meds 
X 
X ------------------------------------------------------------------------------------------------------- X 
Physical exam 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
Height 
X 
Weight 
X 
X 
X 
X 
X 
X 
X 
X 
Performance 
status 
X 
X 
X 
X 
X 
X 
X 
X 
CBC w/diff, plts 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum 
chemistrya 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

NCI Protocol #: 9585 
Version Date: October 8, 2019 
 
75 
 
Screening 
Cycle 1 
Cycle 2 
Cycle 3 and Beyond 
Off Studyf 
Within 
28 
Days 
Within 
14 
Days 
Within 
7 
Days 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
Amylase/Lipasea 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Coagulation 
(PT/PTT/INR)a 
X 
X 
X 
X 
X 
Fasting Lipid 
Panela 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Fasting Serum 
Glucosea 
X 
X 
 
 
 
X 
 
 
 
X 
 
X 
HbA1ca 
X 
X 
B-HCGb 
Xb 
X 
 
 
 
X 
 
 
 
X 
In-Home Glucose 
Monitoringh 
 
X-------------Daily-----------X X-------------Daily-----------X Xh-------------Daily-----------X 
Urinalysisc 
X 
X 
X 
X 
X 
X 
X 
X 
EKGg 
X 
 
 
X 
ECHO/MUGA 
X 
Adverse event 
evaluation 
X ------------------------------------------------------------------------------------------------------- X 
X 
Tumor 
measurementsd 
X 
Tumor measurements are repeated every 8 weeks. Documentation (radiologic) must be 
provided for patients removed from study for progressive disease. 
X 
Radiologic 
evaluation 
X 
Radiologic measurements should be performed every 8 weeks. 
X 
Biopsye 
X 
X 
X 
PBMC Samplee 
X 
X 
X 
A: MLN0128 (TAK-228): Dose as assigned; 3 days on and 4 days off oral starting on Day 2 of every cycle 
B: Ziv-Aflibercept: Dose as assigned; Administered intravenously every 2 weeks (Days 1 and 15 of every cycle) 
a: Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, magnesium, total 
protein, SGOT [AST], SGPT [ALT], sodium will be conducted at baseline and every visit. PT, PTT/INR will be collected at Day 1 of every cycle and off study 
visit. The fasting lipid panel will be collected at baseline, weekly during Cycle 1, Day 1 and Day 15 of Cycle 2 and beyond and off study visit. HbA1c will be 
collected at screening, Cycle 3 Day 1 and every 3 cycles thereafter. Fasting serum glucose will be conducted on Day 1 of all Cycles. 
Amylase/lipase will be obtained at screening, weekly during Cycle 1, every 2 weeks during Cycle 2 and beyond, and Off Study visit. 

NCI Protocol #: 9585 
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76 
 
Screening 
Cycle 1 
Cycle 2 
Cycle 3 and Beyond 
Off Studyf 
Within 
28 
Days 
Within 
14 
Days 
Within 
7 
Days 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
Day 
1 
Day 
8 
Day 
15 
Day 
22 
b: Serum pregnancy test (women of childbearing potential) within 7 days. Serum or urine pregnancy test is required on Day 1 of each cycle. If screening pregnancy 
test is negative, then a Cycle 1 Day 1 pregnancy is not required.  
c: Assess urine protein creatinine (UPC) ratio. If UPC ratio is > 1.0, 24-hour urine collection for protein and creatinine clearance should be obtained. 
d. Tumor assessments will be performed after every two cycles beginning with Cycle 2 and will include CT or MRI and/or PET scan. Whichever modality is 
chosen for tumor assessment, the same modality should be used for serial evaluations of the patient throughout the trial. 
e: Biopsies, and PBMC samples will be taken in the dose expansion cohort only. Baseline biopsy, and PBMC will be obtained within 8 days of C1D1. 
f: Off-study evaluation to be performed within 30 days (+/- 7 days) after discontinuation of study drugs. In addition, for patients who experience significant 
toxicities on study, follow-up should continue until toxicities resolve or are deemed irreversible. 
g. EKG: Performed at screening, as clinically indicated during the study, and during the Off Study Visit. 
h 
 In-home daily fasting glucose monitoring: Patients will be given a glucometer to monitor fasting glucose levels at home collected daily predose on dosing 
days, at approximately the same time each day and will be instructed to notify the study clinician any time the fasting glucose is abnormal (i.e. ≥150mg/dL). In-
home glucose monitoring is not required on days when fasting glucose is measured in the clinic. For further instruction see Section 5.3. 

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11. MEASUREMENT OF EFFECT 
 
Although response is not the primary endpoint of this trial, patients with measurable disease will 
be assessed by standard criteria. For the purposes of this study, patients should be re-evaluated 
every 8 weeks.  In addition to a baseline scan, confirmatory scans will also be obtained 8 weeks 
following initial documentation of an objective response. 
 
11.1 
Antitumor Effect – Solid Tumors 
 
For the purposes of this study, patients should be re-evaluated for response every 8 weeks.  In 
addition to a baseline scan, confirmatory scans should also be obtained 8 (not less than 4) weeks 
following initial documentation of objective response. Scans will include CT or MRI and/or PET 
scan. Whichever modality is chosen for tumor assessment, the same modality should be used for 
serial evaluations of the patient throughout the trial. However, PET CT may be alternated with a 
CT if needed or indicated in a given patient. If a subject has been on study for 12 months the 
staging interval may be increased to re-evaluate for response every 12 weeks (3 cycles). 
 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
(version 1.1) (Eisenhauer et al., 2009).  Changes in the largest diameter (unidimensional 
measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph 
nodes are used in the RECIST criteria. 
 
11.1.1 Definitions 
 
Evaluable for toxicity.  All patients will be evaluable for toxicity from the time of their first 
treatment with MLN0128 (TAK-228) and/or ziv-aflibercept. 
 
Evaluable for objective response.  Only those patients who have measurable disease present at 
baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will 
be considered evaluable for response. These patients will have their response classified 
according to the definitions stated below. (Note: Patients who exhibit objective disease 
progression prior to the end of cycle 1 will also be considered evaluable.) 
 
Evaluable Non-Target Disease Response.  Patients who have lesions present at baseline that are 
evaluable but do not meet the definitions of measurable disease, have received at least one cycle 
of therapy, and have had their disease re-evaluated will be considered evaluable for non-target 
disease.  The response assessment is based on the presence, absence, or unequivocal progression 
of the lesions. 
 
11.1.2 Disease Parameters 
 
Measurable disease.  Measurable lesions are defined as those that can be accurately measured in 
at least one dimension (longest diameter to be recorded) as ≥20 mm by chest x-ray or as ≥10 
mm with CT scan, MRI, or calipers by clinical exam. All tumor measurements must be recorded 
in millimeters (or decimal fractions of centimeters). 
 

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Note:  Tumor lesions that are situated in a previously irradiated area might or might not be 
considered measurable.  If the investigator thinks it appropriate to include them, the conditions 
under which such lesions should be considered must be defined in the protocol. 
 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a lymph 
node must be ≥15 mm in short axis when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm).  At baseline and in follow-up, only the short axis 
will be measured and followed. 
 
Non-measurable disease.  All other lesions (or sites of disease), including small lesions (longest 
diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered 
non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial 
effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses 
(not followed by CT or MRI), are considered as non-measurable. 
 
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts should not 
be considered as malignant lesions (neither measurable nor non-measurable) since they are, by 
definition, simple cysts. 
 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, 
if they meet the definition of measurability described above. However, if non- cystic lesions are 
present in the same patient, these are preferred for selection as target lesions. 
 
Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in 
total, representative of all involved organs, should be identified as target lesions and recorded 
and measured at baseline. Target lesions should be selected on the basis of their size (lesions 
with the longest diameter), be representative of all involved organs, but in addition should be 
those that lend themselves to reproducible repeated measurements.  It may be the case that, on 
occasion, the largest lesion does not lend itself to reproducible measurement in which 
circumstance the next largest lesion which can be measured reproducibly should be selected. A 
sum of the diameters (longest for non- nodal lesions, short axis for nodal lesions) for all target 
lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes are to be 
included in the sum, then only the short axis is added into the sum. The baseline sum diameters 
will be used as reference to further characterize any objective tumor regression in the 
measurable dimension of the disease. 
 
Non-target lesions.  All other lesions (or sites of disease) including any measurable lesions over 
and above the 5 target lesions should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements of these lesions are not required, but the presence, absence, 
or in rare cases unequivocal progression of each should be noted throughout follow-up. 
 
11.1.3 Methods for Evaluation of Measurable Disease 
 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All 
baseline evaluations should be performed as closely as possible to the beginning of treatment 
and never more than 4 weeks before the beginning of the treatment. 
 

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The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam. 
 
Clinical lesions  Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and ≥10 mm diameter as assessed using calipers 
(e.g., skin nodules).  In the case of skin lesions, documentation by color photography, including 
a ruler to estimate the size of the lesion, is recommended. 
 
Chest x-ray Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung.  However, CT is preferable. 
 
Conventional CT and MRI This guideline has defined measurability of lesions on CT scan 
based on the assumption that CT slice thickness is 5 mm or less.  If CT scans have slice 
thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice 
thickness.  MRI is also acceptable in certain situations (e.g. for body scans). 
 
Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which greatly 
impact image quality, lesion conspicuity, and measurement.  Furthermore, the availability of 
MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of 
the scanning sequences used should be optimized for the evaluation of the type and site of 
disease.  Furthermore, as with CT, the modality used at follow-up should be the same as was 
used at baseline and the lesions should be measured/assessed on the same pulse sequence.  It is 
beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence 
parameters for all scanners, body parts, and diseases.  Ideally, the same type of scanner should 
be used and the image acquisition protocol should be followed as closely as possible to prior 
scans. Body scans should be performed with breath-hold scanning techniques, if possible. 
 
PET-CT At present, the low dose or attenuation correction CT portion of a combined PET-CT 
is not always of optimal diagnostic CT quality for use with RECIST measurements.  However, if 
the site can document that the CT performed as part of a PET-CT is of identical diagnostic 
quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be 
used for RECIST measurements and can be used interchangeably with conventional CT in 
accurately measuring cancer lesions over time.  Note, however, that the PET portion of the CT 
introduces additional data which may bias an investigator if it is not routinely or serially 
performed. 
 
Ultrasound  Ultrasound is not useful in assessment of lesion size and should not be used as a 
method of measurement.  Ultrasound examinations cannot be reproduced in their entirety for 
independent review at a later date and, because they are operator dependent, it cannot be 
guaranteed that the same technique and measurements will be taken from one assessment to the 
next.  If new lesions are identified by ultrasound in the course of the study, confirmation by CT 
or MRI is advised.  If there is concern about radiation exposure at CT, MRI may be used instead 
of CT in selected instances. 

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80 
 
 
Endoscopy, Laparoscopy The utilization of these techniques for objective tumor evaluation is 
not advised.  However, such techniques may be useful to confirm complete pathological 
response when biopsies are obtained or to determine relapse in trials where recurrence following 
complete response (CR) or surgical resection is an endpoint. 
 
Tumor markers  Tumor markers alone cannot be used to assess response.  If markers are initially 
above the upper normal limit, they must normalize for a patient to be considered in complete 
clinical response.  Specific guidelines for both CA-125 response (in recurrent ovarian cancer) 
and PSA response (in recurrent prostate cancer) have been published (Gordon et al., 2004; 
Bubley et al., 1999; Scher et al., 2008).  In addition, the Gynecologic Cancer Intergroup has 
developed CA-125 progression criteria which are to be integrated with objective tumor 
assessment for use in first-line trials in ovarian cancer (Vergote et al., 2000). 
 
Cytology, Histology These techniques can be used to differentiate between partial responses 
(PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as 
germ cell tumors, where known residual benign tumors can remain). 
 
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 
mandatory to differentiate between response or stable disease (an effusion may be a side effect 
of the treatment) and progressive disease. 
 
FDG-PET  While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis of 
FDG-PET imaging can be identified according to the following algorithm: 
a. Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD 
based on a new lesion. 
b. No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive FDG- 
PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD.  If 
the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, 
additional follow-up CT scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-
PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, this is not PD. 
c. FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in 
cases where a residual radiographic abnormality is thought to represent fibrosis or 
scarring.  The use of FDG-PET in this circumstance should be prospectively described in 
the protocol and supported by disease-specific medical literature for the indication.  
However, it must be acknowledged that both approaches may lead to false positive CR 
due to limitations of FDG-PET and biopsy resolution/sensitivity. 
 
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater 
than twice that of the surrounding tissue on the attenuation corrected image. 
 
11.1.4 Response Criteria 

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11.1.4.1 
Evaluation of Target Lesions 
 
Complete Response (CR):  Disappearance of all target lesions. Any pathological lymph nodes 
(whether target or non-target) must have reduction in short axis to <10 mm. 
 
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target lesions, 
taking as reference the baseline sum diameters. 
 
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of target lesions, 
taking as reference the smallest sum on study (this includes the baseline sum if that is the 
smallest on study).  In addition to the relative increase of 20%, the sum must also demonstrate 
an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also 
considered progressions). 
 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient increase to 
qualify for PD, taking as reference the smallest sum diameters while on study. 
 
11.1.4.2 
Evaluation of Non-Target Lesions 
 
Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor 
marker level.  All lymph nodes must be non-pathological in size (<10 mm short axis). 
 
Note:  If tumor markers are initially above the upper normal limit, they must normalize for a 
patient to be considered in complete clinical response. 
 
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or maintenance of tumor 
marker level above the normal limits. 
 
Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal 
progression of existing non-target lesions. Unequivocal progression should not normally trump 
target lesion status.  It must be representative of overall disease status change, not a single lesion 
increase. 
 
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the 
treating physician should prevail in such circumstances, and the progression status should be 
confirmed at a later time by the review panel (or Principal Investigator). 
 
11.1.4.3 
Evaluation of Best Overall Response 
 
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started). The patient's best response assignment will 
depend on the achievement of both measurement and confirmation criteria. 
 
 
 

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For Patients with Measurable Disease (i.e., Target Disease) 
 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-CR/Non- 
PD 
No 
PR 
>4 wks. Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR/Non- 
PD/not 
evaluated 
No 
PR 
SD 
Non-CR/Non- 
PD/not 
evaluated 
No 
SD 
Documented at least once >4 
wks. from baseline** 
PD 
Any 
Yes or No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
∗ 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
** 
Only for non-randomized trials with response as primary endpoint. 
*** 
 In exceptional circumstances, unequivocal progression in non-target lesions may be 
accepted as disease progression. 
Note:  Patients with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
reported as “symptomatic deterioration.”  Every effort should be made to document the 
objective progression even after discontinuation of treatment. 
 
For Patients with Non-Measurable Disease (i.e., Non-Target Disease) 
 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
∗ 
‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is 
increasingly used as an endpoint for assessment of efficacy in some trials so to assign 
this category when no lesions can be measured is not advised 
 
11.1.5 Duration of Response 
 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started). 
 

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83 
 
The duration of overall CR is measured from the time measurement criteria are first met for CR 
until the first date that progressive disease is objectively documented. 
 
Duration of stable disease:  Stable disease is measured from the start of the treatment until the 
criteria for progression are met, taking as reference the smallest measurements recorded since 
the treatment started, including the baseline measurements. 
 
11.2 
Antitumor Effect – Hematologic Tumors 
 
Hematologic tumors will not be evaluated in this study. 
 
11.3 
Other Response Parameters 
 
Other response parameters will not be evaluated in this study. 
 
 
12. DATA REPORTING / REGULATORY REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements). 
 
12.1 
Data Reporting 
 
12.1.1 Method 
 
This study will be monitored by the Clinical Data Update System (CDUS) Version 3.0. 
Cumulative protocol- and patient-specific CDUS data will be submitted electronically to CTEP 
on a quarterly basis, either by FTP burst of data or via the CDS web application. 
 
Reports are due January 31, April 30, July 31, and October 31.  Instructions for submitting data 
using the CDUS can be found on the CTEP Web site (http://ctep.cancer.gov/reporting/cdus.html). 
 
Note:  If your study has been assigned to CDUS-Complete reporting, all adverse events (both 
routine and expedited) that have occurred on the study and meet the mandatory CDUS reporting 
guidelines must be reported via the monitoring method identified above. If your study has been 
assigned to CDUS-Abbreviated reporting, no adverse event reporting (routine or expedited) is 
required to be reported via CDUS. 
 
12.1.2 Responsibility for Data Submission 
 
Study participants are responsible for submitting CDUS data and/or data forms to either the 
Coordinating Center or to the Lead Organization on the study quarterly. The date for submission 
to the Coordinating Center or to the Lead Organization will be set by them. CDUS does not 
accept data submissions from the participants on the study.  When setting the dates, allow time 
for Coordinating Center compilation, Principal Investigator review, and timely submission to 
CTEP by the quarterly deadlines (see Section 12.1.1). For trials monitored by CTMS, a quarterly 
report of data will be provided by Theradex to the Coordinating Center. 

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84 
 
 
12.2 
CTEP Multicenter Guidelines 
 
Not applicable to this study. 
 
12.3 
Collaborative Agreements Language 
 
The agent(s) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI 
under a Collaborative Agreement (CRADA, CTA, CSA) between the Pharmaceutical 
Company(ies) (hereinafter referred to as “Collaborator(s)”) and the NCI Division of Cancer 
Treatment and Diagnosis.  Therefore, the following obligations/guidelines, in addition to the 
provisions in the “Intellectual Property Option to Collaborator” 
(http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm) contained within 
the terms of award, apply to the use of the Agent(s) in this study: 
 
1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor 
can Agent(s) be transferred or licensed to any party not participating in the clinical 
study. 
Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and 
shall be maintained as such by the investigators. The protocol documents for studies 
utilizing Agents contain confidential information and should not be shared or distributed 
without the permission of the NCI.  If a copy of this protocol is requested by a patient or 
patient’s family member participating on the study, the individual should sign a 
confidentiality agreement. A suitable model agreement can be downloaded from: 
http://ctep.cancer.gov. 
 
2. For a clinical protocol where there is an investigational Agent used in combination with 
(an)other Agent(s), each the subject of different Collaborative Agreements, the access to 
and use of data by each Collaborator shall be as follows (data pertaining to such 
combination use shall hereinafter be referred to as "Multi-Party Data”): 
 
a. NCI will provide all Collaborators with prior written notice regarding the existence 
and nature of any agreements governing their collaboration with NCI, the design of 
the proposed combination protocol, and the existence of any obligations that would 
tend to restrict NCI's participation in the proposed combination protocol. 
 
b. Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical 
trial by any other Collaborator solely to the extent necessary to allow said other 
Collaborator to develop, obtain regulatory approval or commercialize its own Agent. 
 
c. Any Collaborator having the right to use the Multi-Party Data from these trials must 
agree in writing prior to the commencement of the trials that it will use the Multi-
Party Data solely for development, regulatory approval, and commercialization of 
its own Agent. 
 
3. Clinical Trial Data and Results and Raw Data developed under a Collaborative 

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85 
 
Agreement will be made available to Collaborator(s), the NCI, and the FDA, as 
appropriate and unless additional disclosure is required by law or court order as 
described in the IP Option to Collaborator 
(http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm). Additionally, 
all Clinical Data and Results and Raw Data will be collected, used and disclosed 
consistent with all applicable federal statutes and regulations for the protection of human 
subjects, including, if applicable, the Standards for Privacy of Individually Identifiable 
Health Information set forth in 45 C.F.R. Part 164. 
 
4. When a Collaborator wishes to initiate a data request, the request should first be sent to 
the NCI, who will then notify the appropriate investigators (Group Chair for Cooperative 
Group studies, or PI for other studies) of Collaborator's wish to contact them. 
 
5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the 
guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is 
a DMC for this clinical trial. 
 
6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the 
Group office for Cooperative Group studies or by the principal investigator for non- 
Cooperative Group studies for immediate delivery to Collaborator(s) for advisory review 
and comment prior to submission for publication. Collaborator(s) will have 30 days from 
the date of receipt for review. Collaborator shall have the right to request that publication 
be delayed for up to an additional 30 days in order to ensure that Collaborator’s 
confidential and proprietary data, in addition to Collaborator(s)’s intellectual property 
rights, are protected. 
Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for 
courtesy review as soon as possible and preferably at least three (3) days prior to 
submission, but in any case, prior to presentation at the meeting or publication in the 
proceedings. Press releases and other media presentations must also be forwarded to 
CTEP prior to release. 
Copies of any manuscript, abstract and/or press release/ media presentation should be sent 
to: 
Email:  ncicteppubs@mail.nih.gov 
 
The Regulatory Affairs Branch will then distribute them to Collaborator(s). No publication, 
manuscript or other form of public disclosure shall contain any of Collaborator’s confidential/ 
proprietary information. 

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86 
 
13. STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design/Endpoints 
 
Dr. Suyu Liu (Biostatistics at UT MD Anderson Cancer Center) will perform all statistical 
analyses. The primary objective of this study is to determine the maximum tolerated dose (MTD) 
of the combination of MLN0128 (TAK-228) and ziv-aflibercept in patients with advanced 
cancers refractory to standard therapy. 
 
A standard 3+3 dose-escalation design will be implemented for this phase I portion of the study. 
There will be no intra-patient dose escalation, and no patients will be enrolled in the next dose 
level until the toxicity is fully assessed after the completion of 1 cycle and at least 3 patients 
enrolled at the previous dose level. 
 
Three patients will be treated per dose level. If none of the patients experience a dose-limiting 
toxicity (DLT), the next cohort of three patients will be treated at the next higher dose level. If 1 
of 3 patients treated at a dose experiences a DLT, that cohort will then be expanded to a total of 
six patients. If a DLT occurs in only 1 in 6 of the first dose level, 3 more patients may be 
enrolled at the next higher dose level (i.e., cohort 2). If 2 or more of 6 patients in any cohort 
experiences DLT, the MTD is considered to have been exceeded. No patients may be enrolled in 
the next dose level until 3 patients at the previous dose level have completed at least one cycle of 
therapy. 
 
The software used to compute the statistical designs is Multc Lean v2.1. 
 
Primary Endpoints 
The primary endpoints are safety, tolerability, and determination of DLT and MTD. 
 
Secondary Endpoints 
The secondary endpoints are to determine changes in parameter values compared to baseline, at 
early scans, restaging, and at time of progression will be assessed. 
 
For the expansion cohort only, the secondary endpoints are to determine changes in analyses 
performed on PBMCs and biopsies, and changes of phosphorylation levels of AKT (Ser473) and 
pS6 as well as other biomarkers. 
 
MTD Expansion Cohort 
Once the MTD is determined, the expansion cohort may be opened. The MTD is defined as the 
highest dose level at which no more than 1 of 6 evaluable patients has had a DLT. At the MTD, 
or at Dose Level 3 if no DLTs are observed, six patients should be treated before the dose is 
declared as MTD. 
 
Up to 10 additional patients may be entered at the MTD once it is determined for the purpose of 
collecting additional information on toxicity, tolerability, and biologic effect at that dose. These 
additional 10 patients will be monitored for toxicity. We will emphasize enrollment of pancreatic 
neuroendocrine tumors (pNET) and ovarian carcinoma. There will be discussion between the PI 

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and NCI-CTEP to further expand with additional patients. 
 
Toxicity monitoring for the expansion cohort 
 
We will consider stopping the expansion cohort for excessive toxicity if, among the additional 10 
patients in the expansion cohort (beyond the initial 6), there are > 3/3, 4/5 or 5/9 patients with 
DLT. This would yield approximately 90% likelihood of declaring excessive toxicity for a true 
DLT rate of 0.7 and only approximately 10% likelihood of declaring excessive toxicity for a true 
rate of 0.3. 
 
Analysis of Primary Endpoints 
Data from all patients who receive one or more doses of drug will be incorporated into the final 
safety analysis. To assess clinical safety, all adverse experiences, vital sign measurements, 
clinical laboratory information, radiographic studies and concomitant illnesses will be 
summarized by visit. Adverse experiences and laboratory results will be summarized by severity 
grade (NCI CTCAE version 4.0 until March 31, 2018, and version 5.0 beginning April 1, 2018). 
 
All patients who receive any amount of the study drug will be evaluable for toxicity. The MTD 
is defined as the highest dose level at which no more than 1 of 6 evaluable patients has had a 
DLT. 
 
13.2 
Sample Size/Accrual Rate 
 
Twenty-nine patients were treated in the initial dose escalation dosing schemas. As of 
Amendment 13/ Version 07, three dose levels are included in the revised dose escalation with a 
3+3 design, and there will be a maximum of 18 patients enrolled in the revised dose escalation 
cohorts. At the MTD, an additional 10 patients will be enrolled in the expansion cohort. Prior to 
Amendment 13/ Version 07, there were 29 patients enrolled in the dose escalation. Therefore, the 
maximum accrual is 57 patients (29 + 18 + 10). 
 
Accrual rate: 1-3 patients per month 
 
Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
3 
+ 
6 
= 
9 
Not Hispanic or Latino 
28 
+ 
20 
= 
48 
Ethnic Category: Total of all subjects 
31 
+ 
26 
= 
57 
 
Racial Category 
American Indian or Alaskan Native 
0 
+ 
0 
= 
0 
Asian 
1 
+ 
1 
= 
2 
Black or African American 
2 
+ 
2 
= 
4 

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88 
 
Native Hawaiian or other Pacific 
Islander 
0 
+ 
0 
= 
0 
White 
28 
+ 
23 
= 
51 
Racial Category: Total of all subjects 
31 
+ 
26 
= 
57 
 
 
13.3 
Stratification Factors 
 
None 
 
13.4 
Analysis of Secondary Endpoints 
 
Tumor Response 
The secondary endpoints are to determine changes in parameter values compared to baseline, at 
early scans, restaging, and at time of progression will be assessed. Tumor response will be 
assessed by RECIST (Version 1.1) (Section 11.0). 
 
Evaluation of Tumor Size 
Secondary objectives include getting an early indication of efficacy by assessing tumor size. The 
sum of the largest diameters of the index lesions will be used for tumor size. Tumor size will be 
measured at baseline and at time of best response. For each patient, the % change in tumor size 
from baseline to best response will be computed. We will construct a waterfall plot of these 
values. 
 
Correlative Studies 
For the expansion cohort only, the correlative endpoints are to assess the biological effect of the 
treatment.  Due to the relatively small number of patients, the correlative studies should be 
viewed as exploratory in nature. 
 
Reverse Phase Protein Array (RPPA) 
RPPA will be performed on the tumor biopsies performed at baseline and on Day 8 of cycle 1. 
Total and phosphorylated expression will be determined for several proteins including (but not 
limited to): pAKTSer473, pS6, PTEN, and CD31.  The biomarker of primary interest is phospho- 
AKT. 
 
Immunohistochemistry (IHC): 
IHC will be performed on the tumor biopsies performed at baseline and on Day 8 of cycle 1. 
Total and phosphorylated expression will be determined for several proteins including (but not 
limited to): pAKTSer473, pS6, PTEN, and CD31. The biomarker of primary interest is phospho- 
AKT. 
 
Analyses 
For each marker above, we will assess changes from baseline to cycle 1 day 8 using a paired t- 
test unless the data are clearly not normally distributed in which case we will use a Wilcoxon 
signed rank test. With 10 patients, we can detect a 1-SD mean difference (where SD is the 
standard deviation of the differences) as significant with 80% power using a two-sided 5% alpha. 

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Changes in BF and phospho-AKT are of primary interest and assessment of changes in other 
biomarkers will be considered exploratory. 
 
We will compute the Pearson correlation coefficient between the percent change between 
baseline and cycle 1 day 8 for BF and tumor size. If the data are not normally distributed, we will 
use Spearman’s rank correlation coefficient. With 10 patients, we can detect a correlation 
coefficient of 0.8 or higher as significantly different from zero with 80% power using a two- 
sided 5% alpha. Assessments of correlation between tumor size and changes in other 
biomarkers and among the various biomarker changes will be considered exploratory. 

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REFERENCES 
 
Alvarez EA, Brady WE, Walker J, Rotmensch J, Zhou XC, Kendrick JE, Yamada SD, 
Schilder JM, Cohn D, Harrison CR, Moore, KN, Aghajanian C. Phase II trial of combination 
of aflibercept and temsirolimus in the treatment of recurrent or persistent endometrial 
carcinoma: a gynecologic oncology group study. Gynecologic Oncology. 2012. 
 
Beaudry P FJ, Naumov GN, Wang A, Baker CH, Ryan A, Soker S, Johnson BE, Folkman J, 
Heymach JV. Differential effects of vascular endothelial growth factor receptor-2 inhibitor 
ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: 
implications for use as a surrogate marker of antiangiogenic activity. Clinical Cancer 
Research. 2005; 11:3514-3522. 
 
Bilen MA, Naing A, Hong DS, Falchook GS, Fu S, Wen S, Wheler JJ, Durand JB, Ewer M, 
Kurzrock R. Association between elevated blood pressure and clinical response in patients 
treated with anti-vascular endothelial growth factor (anti-VEGF) therapy in phase I clinical 
trials. J Clin Oncol 30, 2012 (suppl; abstr e13506). 
 
Bubley GJ, Carducci M, Dahut W, Dawson N, et al. Eligibility and response guidelines for 
phase II clinical trials in androgen-independent prostate cancer: recommendations from the 
prostate- specific antigen working group. J Clin Oncol 1999; 3461-3467 
 
Chiang GG, Abraham RT. Targeting the mTOR signaling network in cancer. Trends in 
Molecular Medicine 2007;13(10):433-42. 
 
Dancey JE. Molecular targeting: PI3 kinase pathway. Ann Oncol 2004; 15(Suppl 4):233-239. 
 
Davis DW MD, Abbruzzese, Herbst RS. Surrogate markers in antiangiogenesis clinical trials. 
British Journal of Cancer. 2003; 89:8-14. 
 
Eisenhauer, EA, Therasse P, Bogaerts J, Schwartz LH, et al., New response evaluation criteria 
in solid tumours: revised RECIST guideline (version 1.1). Eur J Ca 2009; 45(2): 228-247. 
 
Fasolo A and Sessa C. mTOR inhibitors in the treatment of cancer. Expert Opin Invest Drugs 
2008; 17(11):1717-1734 
 
Georgakis GV and Younes A. From Rapa Nui to rapamycin: targeting PI3K/Akt/mTOR for 
cancer therapy. Expert Rev Anticancer Ther 2006; 6(1):131-140. 
 
Gordon J, Rustin S, Quinn M, Thigpen T, et al., Re: new guidelines to evaluate the response to 
treatment in solid tumors (ovarian cancer). JNCI J Natl Cacner Inst 2004; 96(6): 487-488. 
 
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144:646-674. 
Heng DYC. Combination therapy in metastatic renal cell carcinoma. The Lancet Oncology 

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2011; 12(7): 613-614. 
Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton 
D, Burova E, Ioffe E, Huang T, Radziejewski C, Bailey K, Fandl JP, Daly T, Wiegand SJ, 
Yancopoulos GD, Rudge JS. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc 
Natl Acad Sci U S A 2002; 99(17):11393-8. 
 
Investigator’s Brochure (VEGF TRAP, AVE0005- Aflibercept), Edition 15 (2017). Sanofi 
R&D, Centre de Recherche Vitry Alfortville, France. 
 
Investigator’s Brochure (MLN0128 (TAK-228)), Edition 10 (2017). Millennium 
Pharmaceuticals, Inc., Cambridge, MA. 
 
Majumder PK, Febbo PG, Bikoff R, Berger R, Xue Q, McMahon LM, Manola J, Brugarolas J, 
McDonnell TJ, Golub TR, Loda M, Lane HA, Sellers WR. mTOR inhibition reverses Akt- 
dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-
dependent pathways. Nat Med. 2004 Jun; 10(6):594-601. Epub 2004 May 23. 
 
Meric-Bernstam F and Mills GB. Mammalian target of rapamycin. Semin Oncol 
2004; 31(Suppl 16):10-17 
 
Naing A, Kurzrock R, Burger A, Gupta S, Lei X, Busaidy N, Hong D, Chen HX, Doyle LA, 
Heilbrun LK, Rohren E, Ng C, Chandhasin C, LoRusso P. Phase I trial of cixutumumab 
combined with temsirolimus in patients with advanced cancer. Clin Cancer Res. 2011 Sep 
15; 17(18):6052-60. 
 
Naing A, LoRusso P, Fu S, Hong DS, Anderson P, Benjamin RS, Ludwig J, Chen HX, Doyle 
LA, Kurzrock R. Insulin growth factor-receptor (IGF-1R) antibody cixutumumab combined 
with the mTOR inhibitor temsirolimus in patients with refractory Ewing’s sarcoma family 
tumors. 
Clin Cancer Res. 2012 May 1; 18(9):2625-31. 
 
Naing A, LoRusso P, Fu S, Hong D, Doyle LA, Phan AT, Habra MA, Kurzrock R. Insulin 
growth factor receptor (IGF-1R) antibody cixutumumab combined with the mTOR inhibitor 
temsirolimus in patients with metastatic adrenocortical carcinoma. British Journal of Cancer 
2013, 1-5. (in print) 
 
Naing A. Overcoming resistance to mTOR inhibition for enhanced strategies in clinical trials. 
Expert Opin Investig Drugs. 2013 Apr 30 
 
Scher HI, Halabi S, Tannock I, Morris M, et al. Design and end points of clinical trials for 
patients with progressive prostate cancer and castrate levels of testosterone: recommendations 
of the prostate cancer clinical trials working group. J Clin Oncol 2008; 26(7): 1148-1159. 
 
Vergote I, Gordon J, Rustin S, Eisenhauer EA et al. Re: new guidelines to evaluate the 
response to treatment in solid tumors (ovarian cancer). JNCI J Natl Cancer Inst 2000; 92(18): 

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1534-1535. 
 
Yao J, Tsuchihashi Z, Paneerselvam A, Winkler R, Bugarini R, Pavel M. Effect of everolimus 
treatment on markers of angiogenesis in patients with advanced pancreatic neuroendocrine 
tumors (pNET): results from the phase III RADIANT-3 study [abstract]. Ann Oncol 2011; 22. 

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APPENDIX A 
PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
100 
Normal, no complaints, no evidence 
of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of disease. 
1 
Symptoms, but ambulatory. 
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work). 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active work. 
2 
In bed <50% of the time. 
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities.  Up and about 
more than 50% of waking hours. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
50 
Requires considerable assistance and 
frequent medical care. 
3 
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours. 
40 
Disabled, requires special care and 
assistance. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
4 
100% bedridden. Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
10 
Moribund, fatal processes 
progressing rapidly. 
5 
Dead. 
0 
Dead. 

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APPENDIX B 
NEW YORK HEART ASSOCIATION (NYHA) CLASSIFICATIONS 
 
Class Functional Capacity 
Objective Assessment 
I 
Patients with cardiac disease but without resulting 
limitations of physical activity. Ordinary physical 
activity does not cause undue fatigue, palpitation, 
dyspnea, or anginal pain. 
No objective evidence of 
cardiovascular disease. 
II 
Patients with cardiac disease resulting in slight limitation 
of physical activity. They are comfortable at rest. 
Ordinary physical activity results in fatigue, palpitation, 
dyspnea, or anginal pain. 
Objective evidence of 
minimal cardiovascular 
disease. 
III 
Patients with cardiac disease resulting in marked 
limitation of physical activity.  They are comfortable at 
rest.  Less than ordinary activity causes fatigue, 
palpitation, dyspnea, or anginal pain. 
Objective evidence of 
moderately severe 
cardiovascular disease. 
IV 
Patients with cardiac disease resulting in inability to carry 
on any physical activity without discomfort. Symptoms 
of heart failure or the anginal syndrome may be present 
even at rest.  If any physical activity is undertaken, 
discomfort is increased. 
Objective evidence of 
severe cardiovascular 
disease. 
Source:  The Criteria Committee of New York Heart Association. Nomenclature and Criteria for 
Diagnosis of Diseases of the Heart and Great Vessels. 9th Ed. Boston, MA:  Little, Brown & 
Co; 1994:253-256. 

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APPENDIX C 
INFORMATION ON POSSIBLE DRUG INTERACTIONS 
 
Appendix C: List of Relevant Cytochrome P450 Inhibitors and Inducers 
Moderate CYP1A2 Inhibitors 
Cimetidine 
methoxsalen  
 
Strong CYP1A2 Inhibitors 
Fluvoxamine 
ciprofloxacin 
 
Clinically Significant Enzyme Inducers 
Carbamazepine 
rifabutin 
St. John’s Wort 
Phenobarbital 
rifampin 
Phenytoin 
rifapentine  
 
 
Source: 
fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteraction
sLabeling/ ucm093664.htm. 
Note that these lists are not exhaustive. 
 
 

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Information on Possible Interactions with Other Agents for Patients and Their 
Caregivers and Non-Study Healthcare Team 
 
The patient 
is enrolled on a clinical trial using the 
experimental agent MLN0128 (TAK-228) and Ziv-Aflibercept.  This clinical trial is 
sponsored by the National Cancer Institute.  This form is addressed to the patient, but 
includes important information for others who care for this patient. 
 
MLN0128 (TAK-228) interacts with many drugs that are processed by your liver.  Because of 
this, it is very important to tell your study doctors about all of your medicine before you start 
this study.  It is also very important to tell them if you stop taking any regular medicine, or if 
you start taking a new medicine while you take part in this study.  When you talk about your 
medicine with your study doctor, include medicine you buy without a prescription at the drug 
store (over-the-counter remedy), or herbal supplements such as St. John’s wort. 
 
Many health care prescribers can write prescriptions. You must also tell your other prescribers 
(doctors, physicians’ assistants or nurse practitioners) that you are taking part in a clinical trial. 
Bring this paper with you and keep the attached information card in your wallet. These 
are the things that you and they need to know: 
 
MLN0128 (TAK-228) interacts with (a) certain specific enzyme(s) in your liver. 
• 
The enzymes in question are strong CYP1A2 inhibitors and CYP inducers. 
• 
MLN0128 (TAK-228) must be used very carefully with other medicines that 
need these liver enzymes to be effective or to be cleared from your system. 
• 
Other medicines may also affect the activity of the enzyme. 
• 
Substances that increase the enzyme’s activity (“inducers”) could 
reduce the effectiveness of the drug, while substances that decrease 
the enzyme’s activity (“inhibitors”) could result in high levels of 
the active drug, increasing the chance of harmful side effects. 
• 
Substances that increase the enzyme’s activity (“inducers”) could 
result in high levels of the active drug, increasing the chance of 
harmful side effects, while substances that decrease the enzyme’s 
activity (“inhibitors”) could reduce the effectiveness of the drug. 
• 
MLN0128 (TAK-228) is considered an “inducer/inhibitor” of the 
enzyme, meaning that it can affect the levels of other drugs that are 
processed by that enzyme. This can lead to harmful side effects 
and/or reduce the effectiveness of those medications. 
• 
You and healthcare providers who prescribe drugs for you must be careful 
about adding or removing any drug in this category. 
• 
Before you start the study, your study doctor will work with your regular 
prescriber to switch any medicines that are considered “strong CYP1A2 
inhibitors and CYP inducers”. 
• 
Your prescribers should look at this web site  
http://medicine.iupui.edu/clinpharm/ddis/table.aspx or consult a medical 
reference to see if any medicine they want to prescribe is on a list of drugs to 
avoid. 

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• 
Please be very careful!  Over-the-counter drugs have a brand name on the 
label—it’s usually big and catches your eye. They also have a generic name—
it’s usually small and located above or below the brand name, and printed in 
the ingredient list. Find the generic name and determine, with the pharmacist’s 
help, whether there could be an adverse interaction. 
• 
Be careful: 
• 
If you take acetaminophen regularly: You should not take more 
than 4 grams a day if you are an adult or 2.4 grams a day if you are 
older than 65 years of age. Read labels carefully!  Acetaminophen 
is an ingredient in many medicines for pain, flu, and cold. 
• 
If you take herbal medicine regularly: You should not take St. 
John’s wort while you are taking MLN0128 (TAK-228). 
 
Other medicines can be a problem with your study drugs. 
• Concomitant administration of any proton pump inhibitor (PPI) is not permitted during 
the study. Patients receiving PPI therapy before enrollment must stop using the PPI for 
7 days before their first dose of study drugs. Examples of PPIs include omeprazole, 
esomeprazole, pantoprazole, lansoprazole, and rabeprazole. 
• See restrictions that apply to the use of histamine H2 receptor antagonists, neutralizing 
antacids, calcium and anti-gas preparations. 
• You should check with your doctor or pharmacist whenever you need to use an over-
the-counter medicine or herbal supplement. 
 
• Your regular prescriber should check a medical reference or call your study doctor 
before prescribing any new medicine for you. Your study doctor’s name is  
__________________________________ 
and he or she can be contacted at  _____________________________ 

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INFORMATION ON POSSIBLE DRUG INTERACTIONS  
You are enrolled on a clinical trial using the experimental agent 
__________________.  This clinical trial is sponsored by the NCI.  
__________________ interacts with drugs that are processed by your 
liver.  Because of this, it is very important to: 
 Tell your doctors if you stop taking regular medicine or if you start 
taking a new medicine. 
 Tell all of your prescribers (doctor, physicians’ assistant, nurse 
practitioner, pharmacist) that you are taking part in a clinical trial. 
 Check with your doctor or pharmacist whenever you need to use 
an over-the-counter medicine or herbal supplement. 
 
 
 
 
__________________ interacts mainly with a specific liver enzyme 
called CYP1A2 and additionally with CYP3A4, and must be used very 
carefully with other medicines that interact with this enzyme.  
 Before you start the study, your study doctor will work with your 
regular prescriber to switch any medicines that are considered 
“strong inducers/inhibitors of CYP3A4, 2C9, or 2C19.” 
 Before prescribing new medicines, your regular prescribers should 
go to http://medicine.iupui.edu/clinpharm/ddis/table.aspx for a 
list of drugs to avoid, or contact your study doctor.   
 Your study doctor’s name is _____________________________ 
and can be contacted at _________________________________.  

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APPENDIX D 
PATIENT’S MEDICATION DIARY - AGENT MLN0128 (TAK-228) 
 
Today’s date _____________________ 
 
Patient Name_____________________(initials acceptable)  
 Patient Study ID __________________________ 
INSTRUCTIONS TO THE PATIENT: 
1. Complete one form for each cycle of treatment. 
2. Your dose = ____ mg.  Take ____ 1 mg and/or  ____ 3 mg capsules. You will take MLN0128 (TAK-228) capsules via mouth for 3 days on and 4 days 
off starting on Day 2 of every cycle. Follow the dose selected by your physician. You should take MLN0128 (TAK-228) with a full glass of water. You 
should not eat for 2 hours before and 1 hour after you take MLN0128 (TAK-228) 
3. Record the date, the number of capsules that you took, and when you took them 
4. If you have any comments, please record them in the Comments column and notify the study coordinator or research nurse. 
5. Please bring this form and your bottles of MLN0128 (TAK-228) capsules when you return for each appointment. 
6. If you miss a dose, take it as soon as you remember. If it is almost time for your next dose, skip the missed dose and take the medicine at the next 
regularly scheduled time. Do not double the doses to make up the missed dose. 
7. If you vomit after taking MLN0128 (TAK-228), please record in the comments section and do not take another dose until the next day’s dose. 
8. Please record your daily glucose reading and daily blood pressure (BP). 
 
Day 
Date 
Time of dose 
# of Capsules taken 
Glucose 
BP 
Comments 
1 mg 
3 mg  
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 

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Day 
Date 
Time of dose 
# of Capsules taken 
Glucose 
BP 
Comments 
1 mg 
3 mg  
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
25 
26 
27 
28 
 
Patient’s signature: ________________________ 
Date: ___________________ 

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Physician’s Office will complete this section: 
1. Date patient started protocol treatment_______________________________________________________ 
2. Date patient was removed from study_______________________________________________________ 
3. Patient’s planned total daily dose___________________________________________________________ 
4. Total number of capsules taken this month___________________________________________________ 
5. Physician/Nurse/Data Manager’s Signature __________________________________________________ 
 

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APPENDIX E 
GLUCOSE MONITORING INSTRUCTIONS 
 
In-Home Daily Fasting Glucose Monitoring for 2013-0665 
 
• The level should be collected daily, predose on dosing days, and at approximately the 
same time each day. 
 
• Collect FASTING in the am, prior to taking your MLN1028 pill. 
 
• Record the value in your drug diary.   
o If >150, call the triage nurse (713- 563-1930) 
o If the triage nurse is not available, proceed to your local ER. 
 
• Bring your glucometer back with your drug diary when you see the MD. 
 
• Collect daily, for 2 months, at the same time.  After 2 months your doctor will decide if 
the frequency can be reduced to once weekly. 
Patients will continue to notify the clinic (Triage nurse 713- 563-1930) of fasting blood glucose 
levels that exceed 150 mg/dL and, if blood glucose levels are not well controlled, or if the patient 
requires either oral hypoglycemic agents or insulin to control blood glucose levels, then the 
frequency of in-home testing of FBG levels will be reinstated to daily 

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APPENDIX F 
BIOMARKER ASSAY CHARACTERIZATION 
 
Study Checklist for CTEP-Supported Early Phase Trials with BIOMARKER ASSAYS 
 
INSTRUCTIONS: For INTEGRAL assay, respond to Items 1-7. For 
INTEGRATED assay, respond to Items 1-3 and 5-7. (In lieu of completing items 5-6, the 
CTEP assay templates available at  
http://ctep.cancer.gov/protocolDevelopment/default.htm#ancillary_correlatives may be 
utilized.) 
 
Please submit a response to each of the criteria below and complete one Study Checklist for 
each Biomarker endpoint. 
 
1. 
Markers: 
 
Primary markers: Akt, phospho-Akt (p-Akt) (Thr308), p-Akt (Ser473), CD31, and PTEN. 
 
Secondary markers: p-4E-BP1 (Thr37/46), p-4E-BP1 (Ser65), GSK3alpha/beta (Ser21/9), 
IGF-1Rbeta, IRS1, p-MAPK (Thr202/204), p-MEK1/2 (Ser217/221), p-mTOR (Ser2448), p- 
p27 (Thr157), p-p27 (Thr198), p-p38 MAPK (Thr180/182), PCNA, PI3K p85, PI3K p110 
alpha, p-S6 (Ser235/236), p-S6 (Ser240/244), S6K1, p-S6K1 (Thr389), and p-TSC2 
(Thr1462). 
 
2. 
For an integral or integrated assay, indicate the role(s) of the biomarker assay in the 
trial: N/A 
A. Eligibility criterion – N/A 
B. Assignment to treatment – N/A 
C. Stratification variable – N/A 
D. Risk classifier or score – N/A 
E.  Other (describe in detail): 
 
3. 
Identify the specific individual(s) and laboratory(ies) who are being considered 
for conducting the assay(s) for the trial. 
 
Specimen collection and handling: Argun Akcakanat, MD, PhD. RPPA analysis: MD 
Anderson Cancer Center RPPA Core Facility. 
 
4. 
Integral laboratory assays used for clinical decision-making must be performed in a 
CLIA-certified facility.  Provide the lab’s CLIA number that is performing the 
integral biomarker study(ies) and the expiration date of the certificate. N/A 
 
5. 
Describe the assay: 
 
A. Specify the analyte(s), technical platform, and sources of assay components 
(e.g., reagents, chips, and calibrators). 
 
RPPA arrays will be performed according to SOP at MD Anderson RPPA Core Facility. 

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Frozen samples will be thawed at room temperature and heated at 50°C for 15-20 
minutes. Tumor lysates will be diluted in five 2-fold serial dilutions (1:1, 1:2, 1:4, 1:8, 
and 1:16) in dilution buffer (lysis buffer containing 1% SDS) in 96-well plates. Equal 
volumes of 80% glycerol and 2x PBS will be added to each well. Samples will be 
transferred to 384-well master plates for printing. Plates will be sealed and stored at -
80°C until use. 
 
An Aushon 2470 Arrayer (Aushon BioSystems) will create 1056 spot arrays on 
nitrocellulose-coated slides (Grace Biolab) from serial dilutions. Total array spots will be 
arranged on each slide including the spots corresponding to positive and negative controls 
prepared from mixed cell lysates or dilution buffer, respectively. 
 
Every antibody in the panel was subjected to a stringent validation procedure. Only 
antibodies with a single or dominant band and no nonspecific binding on western blotting 
were further assessed by direct comparison to RPPA. Antibodies that recognized known 
cell characteristics, such as size variants due to cleavage, mutations or deletions were 
acceptable. To validate antibodies against phosphorylated epitopes, cell lines were 
stimulated (e.g. growth factors) or inhibited by specific inhibitors to generate 
phosphorylated and nonphosphorylated forms of a protein. Also, cell lines that were 
genomically altered, such as DNA transfected and overexpressing or short interfering 
RNA transfected and inhibited, could be used for validation. If antibodies passed the 
above criteria, results of RPPA and western blotting had to be parallel. Only antibodies 
with a Pearson correlation coefficient greater than 0.7 between RPPA and western 
blotting as well as wide dynamic range will be used. 
 
Staining will be accomplished by a Dako - Autostainer Link 48 (Dako North America, Inc., 
Carpinteria, CA). Slides will be precleared with ReBlot Plus Mild Antibody Stripping 
Solution (EMD Millipore, Billerica, MA) in PBS containing 0.1% Tween-20 for 30 minutes. 
The blocking and amplification reagents are provided in Catalyzed Signal Amplification 
System (CSA, Dako). Following TBS-T rinse, slides will be blocked for peroxidase, avidin, 
biotin, and proteins 5 minutes each and followed by TBS-T washes. Slides will be incubated 
with primary antibody (Appendix 1) for 20 minutes, TBS-T washed and incubated with the 
secondary antibody (Vector Laboratories, Inc., Burlingame, CA) for 20 minutes. 
Antibodies will be diluted in Antibody Diluent with Background Reducing Components 
(Dako). After TBS-T wash, slides will be subjected to amplification reagents, strepavidin- 
biotin complex, amplification reagent and streptavidin-HRP. For signal detection, 
diaminobenzidine (DAB) will be added to slide and after 3 minutes of incubation, staining 
reaction will be stopped by washing the slide with distilled water. The slides will then be 
air-dried. 
 
The slides will be scanned on a Canon flatbed scanner at 1200 dpi in grayscale, and saved 
as a 16-bit tiff file. Spot intensities will be determined as mean net values, in which the local 
background intensity will be subtracted, by reading tiff images on Array-Pro Analyzer 
software (Media Cybernetics, Inc., Rockville, MD). A representative natural 
logarithmic value of each sample curve on the slide (curve average) was then used as a 
relative quantification of the amount of each protein in each sample. 

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Each dilution curve will be fitted with a logistic model written in the language R 
(Supercurve Fitting, developed by the Department of Bioinformatics and Computational 
Biology in MD Anderson Cancer Center, 
“http://bioinformatics.mdanderson.org/OOMPA”). Briefly, this fits a single curve using all 
the spot intensities of all the samples on a slide with the signal intensity as the response 
variable and the dilution steps are independent variable. Supercurve predicts that each 
antibody has unique binding properties. The fitted curve is plotted with the signal 
intensities – both observed and fitted - on the y-axis and the log2-concentration of proteins 
on the x-axis for diagnostic purposes. This fitted logistic curve is called “supercurve.” 
Goodness of fit of supercurve is determined by calculating a mean R2 of the linear portion 
of the curve (trimmed mean) and color scale mapping of residuals R2 for each spot 
intensity. In addition to visual inspection of slides for evenness of staining, sufficient signal 
intensity, and titration of diluted spots, quantitation data provides strict measures. 
Good quality slides have a trimmed mean R2 over 0.5 and a residuals R2 map of at least 
50% of spots in the acceptable range. 
 
At the final step, the protein concentrations of each set of slides will then be normalized by 
median polish, which was corrected across samples by the linear expression values using 
the median expression levels of all antibody experiments to calculate a loading correction 
factor for each sample. 
 
B. Describe the specimens, and anticipated methods for specimen acquisition, fixation 
or stabilization, and processing.  Provide justification for the timing of specimen 
collection. 
 
Sample acquisition and initial processing: Whole blood will be collected by 
Investigational Cancer Therapeutics (ICT) personnel, and patients will be transferred 
to the Department of Interventional Radiology. Whole blood for extraction of 
peripheral mononuclear cells (PBMC) will be collected in an 8 ml CPT (cell preparation 
tube) with sodium citrate (BD, Becton, Dickinson and Company). Whole blood for 
preparation of platelet rich plasma will be collected in CTAD (buffered sodium citrate 
theorphylline adenosine dipyridamole) tube. The transfer of CPT and CTAD tubes to Dr. 
Funda Meric-Bernstam’s lab and initiation of sample processing will begin within 30 
minutes. Detailed protocols for PBMCs and platelet rich plasma are provided as 
Appendices 2 and 3, respectively. Once preparation is completed, at the final step both 
PBMC and platelet rich plasma samples will be snap- frozen in liquid nitrogen and 
stored at -80°C. Tumor biopsies will be done by interventional radiology and biopsies 
will be snap-frozen in liquid nitrogen according to the protocol in Appendix G. After 
being transferred to Dr. Funda Meric-Bernstam’s lab, tumor samples will also be stored 
at -80°C until use. 
 
Sample processing for RPPA: PBMC pellets will be resuspended in RPPA lysis buffer (1% 
Triton X100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EGTA, 100 mM NaF, 10 mM 
NaPPi, 10% glycerol, 1 mM Na3VO4, cOmplete Protease Inhibitor Cocktail Tablets 
(Roche Diagnostics Corporation, Indianapolis, IN) containing 0.25% sodium deoxycholate. 

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Samples will be lysed on ice and following centrifugation for 10 min at 4C at 16000 xg in a 
microcentrifuge, supernatant will be transferred to a clean microfuge tube. Protein 
concentration will be determined by bicinchroninic acid (BCA) assay (Pierce 
Biotechnology, Rockford, IL). Protein samples will be denatured by adding 4x SDS sample 
buffer (35% glycerol, 8% SDS, 250 mM Tris-HCl, pH 6.8, 10% 2-mercaptoethanol). Final 
PBMC sample concentration will be adjusted to 3 μg/μl. Lysates will be boiled, aliquoted 
and stored at -80°C until use. 
 
Platelet rich plasma pellets will be resuspended in RPPA lysis buffer. Samples will be lysed 
on ice and following centrifugation for 10 min at 4°C at 16000 x g in a microcentrifuge, 
supernatant will be transferred to a clean microfuge tube. Protein concentration will 
be determined by BCA assay. Protein samples will be denatured by adding 4x SDS 
sample buffer. Final PBMC sample concentration will be adjusted to 2 μg/μl. Lysates 
will be boiled, aliquoted and stored at -80°C until use. 
 
Tumor samples will be transferred to a ceramic bead tube and RPPA lysis buffer will be 
added. Samples will be homogenized using a table top tissue homogenizer (Precellys 24, 
Bertin Technologies, Montigny-le-Bretonneux, France) at 4°C. Following centrifugation for 
10 min at 4°C at 16000 x g in a microcentrifuge, supernatant will be transferred to a clean 
microfuge tube. Protein concentration will be determined by BCA assay. Protein samples 
will be denatured by adding 4x SDS sample buffer. Final PBMC sample concentration will 
be adjusted to 1.5 μg/μl. Lysates will be boiled, aliquoted and stored at -80°C until use. 
 
C. Describe the scoring procedures and type of data to be acquired: 
 
Samples are quantitative/continuously distributed in log2 and fold change from baseline 
will be reported as the primary endpoint for signaling markers indicated in the protocol. 
 
6. 
Provide data on the analytical performance of the assay. 
A. For in vitro tests, describe the current status of studies defining the accuracy, 
precision, reportable range, reference ranges/intervals (normal values), turn-around 
time and failure rate of the assay as it is to be performed in the trial. Describe the 
use of positive and negative controls, calibrators, and reference standards for 
clinical assays.  Describe any critical preanalytic variables. For guidance on 
regulatory requirements for laboratory assays please visit: 
 
RPPA is a well-established assay, supported by several published articles. Appendix 4 lists 
selected studies where samples were processed in MD Anderson RPPA Core Facility. This 
list also includes studies completed in Meric-Bernstam lab. 
 
The relatively slow growing component of the assay is the antibody list. This is because of 
the stringent conditions defined to validate RPPA quality antibodies. The validated 
antibodies are defined as: only antibodies with a single or dominant band and no 
nonspecific binding on western blotting were further assessed by direct comparison to 
RPPA (Figure 1). Antibodies that recognized known cell characteristics, such as size 

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variants due to cleavage, mutations or deletions were acceptable. To validate antibodies 
against phosphorylated epitopes, cell lines were stimulated (e.g. growth factors) or 
inhibited by specific inhibitors to generate phosphorylated and nonphosphorylated forms 
of a protein. Also, cell lines that were genomically altered, such as DNA transfected and 
overexpressing or short interfering RNA transfected and inhibited, could be used for 
validation. If antibodies passed the above criteria, results of RPPA and western blotting 
had to be parallel. Only antibodies with a 
 
 
 
Figure 1. RPPA 
slide probed with 
validated 53BP1 
antibody (upper 
panel), 
supercurve 
fitting and 
trimmed mean 
R2 (middle 
panel), and color 
scale mapping 
(lower panel). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pearson correlation coefficient greater than 0.7 between RPPA and western blotting as 

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well as wide dynamic range will be used. As of October 3, 2013, there are 204 antibodies 
included in the standard antibody list of the RPPA Core Facility. The validation data of 
the antibodies listed in “1. Markers,” are provided in Appendix 5. RPPA antibody panel 
is dynamic and some antibodies are removed from this panel in time, when they no 
longer meet the expected criteria. The major reasons to stop using an antibody are: (1) 
the antibody is no longer available, it is discontinued by the company, (2) lot number is 
changed and the new lot does not pass validation, (3) red blob antibodies are removed 
from the standard list for tissue samples. ‘Red blob antibody’ recognizes unidentified 
‘damaged’ components in addition to its specific protein. The ‘damaged’ components 
were observed only in certain tissue samples causing very high positive signals to 
interfere with the network display. 
 
The RPPA Core Facility has a two Aushon 2470 arrayers for printing of up to 100 slides 
per run with several automated runs feasible in a day. There are two Dako - Autostainer 
Link 48 staining systems that probes each slide with a different antibody. Each autostainer 
is capable of staining approximately 60 slides per day under conditions that are specific 
for each individual antibody. 
 
Lysates will be serial diluted to define the linear range of each antigen-antibody reaction. 
Protein extraction methods of tumor tissue, PBMC and platelet rich plasma lysates show 
a couple of minor differences. First, preliminary studies showed that adding 0.25% 
sodium deoxycholate to RPPA lysis buffer for PBMCs increased the signal intensity 
in western blotting, whereas it had no significant effect on the signal intensity in RPPA. 
Second, final protein concentration is different for these three samples. If we describe 
the level of blood contamination of these three types of samples, the ranking would be 
PBMC > platelet rich plasma > tumor. Protein quantitation assays are colorimetric and 
contaminants result in a false increase in optic density readings. Thus result in a 
discrepancy between reported and real protein concentrations. Preliminary studies 
comparing signal intensities in western blotting and RPPA experiments showed that a 
correction in the final protein concentration was the most easy and reliable way to 
overcome this discrepancy. 
 
Data will be collected and analyzed using quantification software Array-Pro Analyzer. 
This 
software has multiple features including automated spot identification, 
background correction, controlling for location, serial dilution-signal intensity curve 
construction and concentration determination. The values derived from the slope and 
intercept will be expressed relative to standard control cell lysates or control peptides on 
the array. Lastly, Supercurve application is collaboration between the RPPA Core 
Facility and the Department of Bioinformatics, which has updated several times to 
answer the needs of the users, particularly providing more reliable data. 
 
B. If the assay will be performed at more than one site, describe how inter-
laboratory variability in the measurements listed in 5A above will be assessed. 
Describe how these sources of variation will be minimized to maintain 
performance at all sites within acceptable limits and to prevent drift or bias in 
assay. N/A 
 

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7. 
Provide data on the clinical utility of the integral/integrated assay as it will be used in 
the trial: 
A. Provide background information that justifies the use of this assay result as a 
marker for this trial. State the hypothesis and rationale for utilizing the biomarker, 
with supporting preclinical and clinical data, when available.  For example, if the 
integral marker will be used as a stratification or treatment-determining variable, 
data supporting its prognostic or predictive association with a main trial endpoint 
should be described or referenced. 
B. Describe the expected distribution of the biomarker in the study population. 
Justify the number of patients and specimens to determine feasibility and to 
demonstrate that the studies are likely to produce interpretable results. 
C. If cutpoints will be used, specify the cutpoint(s) and describe how these will be 
used in the trial.  Provide the rationale for the cutpoint(s) selected. What 
proportion of subjects is expected to have values above and below the proposed 
assay value cutpoints?  What magnitude of effect (e.g., treatment benefit) or 
outcome (e.g., prognosis) is expected for patients with assay results above and 
below the proposed cutpoint(s)? 
D. Describe the conditions under which treating physicians and or patients will be 
able to access the biomarker assay results. 
In the context of this study, patients in dose expansion will undergo serial biopsies of 
tumors to characterize the effects that MLN0128 (TAK-228) and Aflibercept has upon 
the biology of both the tumor and the stroma. Tumor biopsies will be obtained through 
Interventional Radiology if considered minimal surgical risk. The timing of biopsies is 
defined to allow analysis of the effects of MLN0128 (TAK-228) and Aflibercept upon 
the tumor and stroma in dose expansion before treatment, after treatment with 
MLN0128 (TAK-228) and Aflibercept, and upon progression on both agents. This plan 
will allow the discovery of possible predictive biomarkers that can be queried in future 
trials with this combination. 
In collaboration with Dr. Funda Meric-Bernstam, we will evaluate protein expression (both 
total and phosphorylated proteins) by RPPA and IHC staining on slides from tumor 
samples. Expression of PI3K/mTOR signaling markers and angiogenesis will be assessed 
(Figure 1). Microvessel density will also be evaluated in the tumor tissue using IHC and 
correlated with clinical tumor response and RPPA analyses. Pre- and post-treatment 
biopsies will be evaluated by H&E. Treatment-induced apoptosis will be assessed with 
IHC for cleaved caspase 3 and proliferation will be assessed by Ki-67. 

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Figure 1. Examples of positive and negative staining PI3K/mTOR markers by IHC.  
IHC for PTEN, pAkt Ser473, p4E-BP1 Thr70, pS6 Ser235/236, pS6 Ser240/244 and Ki-
67. 
Breast cancer samples with positive immunostaining are shown in the top panel, and 
samples with negative or low immunostaining are shown in the bottom panel. Data 
provided by Dr. Funda Meric-Bernstam. 
 
In the event that tissue material is limited, we will give priority to RPPA analysis as 
RPPA is an extremely effective and sensitive method for measuring multiple proteins in 
cancer cells.  This technique can be applied to minute amounts of material.  Dr. Funda 
Meric- Bernstam and Dr. Gordon Mills have established this method, and we are 
confident that we can use this technology to measure all the markers of relevance to 
this study.  However, when it is possible to perform both RPPA and IHC for the same 
proteins, the results from one will validate the results from the other. 
 
Reverse phase protein array (RPPA) is a recently developed quantitative assay that 
analyzes nanoliter amounts of sample for potentially hundreds of proteins. This antibody- 
based assay determines levels of protein expression, as well as protein modifications such 
as phosphorylation, cleavage, and fatty acid modification. RPPA allow concordant 
interrogation of multiple signaling molecules and their functional status. In essence, the 
RPPA study has major strengths in identification and validation of cellular targets, 
characterization of signaling pathways and networks, as well as determination of on 
and off target activity of novel drugs. The integrated information will display the 
potential therapeutic targets or biomarkers to accurately predict or rapidly define 
intracellular signaling networks and functional outcomes affected by therapeutics. 
Thus, the RPPA approach provides a method to assess multiple markers in a global 
manner. Linking robust pathway mapping approaches to molecular therapeutics should 
provide an expanding repertoire of validated biomarkers and targeted therapeutics for 
clinical evaluation. Others have shown the benefit of measuring a functional subset of 
proteins using a RPPA in an exploratory manner in the expansion cohort of a trial 
using the combination of sorafenib and bevacizumab2. This subset included proteins in 
the canonical PI3K/AKT/mTOR pathway as well as other signaling proteins. We 
hypothesize that inhibition of the PI3K/AKT/mTOR pathway at a protein level will be 
predictive of response to therapy with MLN0128 (TAK-228). Patients in stage 2 will 
undergo serial biopsies prior to initiation of therapy and after two weeks of single agent 

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MLN0128 (TAK-228) to characterize the effects that MLN0128 (TAK-228) has upon 
the biology of both the tumor and the stroma. Current validated antibodies in the 
RPPA include MEK1/2 (S217/221), AKT (S473, T308), PI3K, p70S6K (T389), MAPK1/2 
(T202/204), GSK3beta(S21/S9), p27 (T157, T198), p38MAPK (T180/182), rS6K 
(S235/236, 
S240/244), and 4E-BP1 (S65). On-study biopsy (D1) can occur up to one week prior to 
initiation of therapy. We estimate that 10 patients per arm will provide power for 
exploratory translational analyses examining target presence, activation, modulation, and 
relationship to clinical outcome. This plan will allow the discovery of possible predictive 
biomarkers that can be queried in future trials with this combination. To be conservative, 
the sample size required was calculated under the assumption that a Bonferroni 
adjustment is required for the 10 primary evaluations to be performed. Thus, requiring 
a two-tailed p-value <0.005 (=0.05/10) for each test to be considered statistically 
significant will be used to estimate the desired number of subjects. In practice, a Hochberg 
adjustment will be used to evaluate the significance of the multiple comparisons, 
which is not as stringent as a Bonferroni adjustment, but will still maintain an overall 
0.05 significance level for the comparisons performed. Furthermore, since this study is 
mainly pilot in intent, the findings will be reported in that context. With 10 patients in 
a cohort, for each such comparison  after single and combination drug administration, 
there is 80%  power to detect a difference from baseline equal to 1.5 standard deviation 
of the change from baseline for each of the 10 parameters, with an overall alpha=0.05 
as stated above. This calculation assumes that a paired t-test will be used to test the 
difference in time for each parameter. If these changes are not normally distributed 
(p<0.05 by Shapiro-Wilks test), then a Wilcoxon signed rank test will be used instead of 
a t-test. In addition, a paired t-test or Wilcoxon signed rank test will be used to evaluate 
the significance of changes for each correlative end point. 
Previous clinical trials have demonstrated the feasibility of proteomics analysis on clinical 
samples associated with targeted agents in these populations. We expect to identify 1-2 
predictive markers to help design a biomarker driven phase 2 trial at the completion of 
this study. There are potential pitfalls to this study including failure to identify a 
predictive marker from our primary endpoint measurements. We will analyze more than  
100 proteins in excess of those identified as our primary endpoints in the specific aims. 
While the statistics were powered for analysis of these primary endpoints, deeper analysis 
of the proteomic datasets in an exploratory fashion may provide other markers of 
interest to future trials. Given the datasets available from previous clinical trials that 
demonstrated some efficacy for TORC1 inhibition or single agent bevacizumab, a 
comparative analysis with the data obtained in this trial might yield important clues as 
to inherent resistance to the combination. 

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APPENDIX G 
COLLECTION AND PROCESSING OF TUMOR BIOPSY AND 
SAMPLES 
 
MATERIALS & EQUIPMENT REQUIRED 
• Liquid nitrogen (LN2) 
• Sterile, disposable tweezers (Fisher #NC9566754) 
• Two cryogenic vials per biopsy sample (one cryogenic vial), placed on dry ice in a 
small insulated bucket (Fisher #72.694.006) 
• One cryogenic vial per biopsy sample filled with 2mL 10% formalin 
• 50-mL conical tubes (Fisher #14-432-22) 
• Computer with specimen processing data sheet 
• 3 sample ID labels per biopsy (2 ID labels if collecting at MDACC) with date and 
time function 
• -80°C freezer 
• Thermoflask cooler 
• Sterile, disposable razor blades 
 
PROCEDURES 
1. Print out labels for each biopsy in advance. The labels should include protocol #, 
patient ID, specimen type, dose level, cycle and time-point of pre-dose or post-dose. 
2. The specimen will be divided into 2 portions, one of which will be snap frozen in 
LN2 and one of which will be fixed in 10% formalin and sent to the respective 
MDACC laboratories. 
3. Send an email in advance to advice recipients (e.g., laboratory designees, study 
coordinator, etc.) of scheduled biopsy and the estimated specimen processing time 
and estimated shipping time. 
4. Prepare and bring necessary lab supplies and reagents including a Thermoflask cooler 
containing approximately 2/3 full LN2, tweezers, sterile razor blades, cryogenic vials 
on dry ice, one cryogenic vial filled with 2mL 10% formalin, lab notes and labels. 
5. Arrive at the surgery unit about 10 minutes ahead of the scheduled procedure giving 
time to set up lab supplies, reagents ready for biopsy specimen processing and data 
recording on time. 
6. Complete specimen processing documentation sheet to record time for each 
processing step as listed on the label sheet and batch record, including: 
 
a) Local anesthesia administered.  Please note: there is a possibility that 
epinephrine in small quantities may interfere with the assay. Therefore, the 
local anesthesia used for tumor biopsies should be only lidocaine whenever 
possible and case report forms should specify what agents (i.e., lidocaine alone 
or lidocaine plus epinephrine) were used for each biopsy procedure. 
b) Skin incision 
c) Guide needle introduced 
d) Guide needle placement confirmed 
e) biopsy needle introduced 
f) portion of biopsy specimen transferred into tube(s) for LN2 

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g) portion of biopsy specimen transferred into tube containing 10% formalin 

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h) Return to the laboratory specimen processing site 
i) Cryopreserved biopsies placed at -80°C. 
 
7. For biopsy specimens that are to be snap frozen in LN2, transfer specimen into a labeled 
cryogenic vial that is pre-chilled in dry ice, and immediately drop the vial with 
specimen into LN2. Dispose of the tweezers into medical waste for sharps. 
 
8. For biopsy specimens that are to be fixed in formalin, transfer specimen into a 
labeled vial containing 2mL 10% formalin. Dispose of the tweezers into medical 
waste for sharps. 
 
9. Transport all biopsy specimens with double container from surgical suite to 
sample processing lab. 
 
10. Transfer all cryopreserved biopsy specimens from LN2 to -80°C and store until ready 
to ship or process. 

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APPENDIX H 
PROCEDURES FOR ISOLATING PERIPHERAL BLOOD 
MONONUCLEAR CELLS (PBMC) AND PLASMA 
 
Isolation of PBMCs: 
 
Equipment 
• Two BD Vacutainer CPT Cell Preparation Tube with EDTA 
• 50 ml size plastic conical centrifuge tubes with caps 
• Pasteur pipettes 
• Temperature-controlled centrifuge with swinging bucket rotor and adapters for 16 X 125 
mm tube size 
• Coulter counter or hemocytometer 
 
Reagents 
• Phosphate Buffered Saline (PBS) without Ca++ or Mg++ 
• 0.4% Trypan blue 
 
Procedures 
1. The BD Vacutainer CPT Tube with EDTA should be used at room temperature (18-
25oC) and properly labeled for patient identification and all related information. 
2. Collect 8 mL of blood each into two Vacutainer CPTs.  Invert tubes approximately 8-
10 times to ensure that the whole blood is mixed thoroughly with the anticoagulant. 
3. Store tubes upright at room temperature until centrifugation. For best results, 
centrifuge the tubes within 30 minutes after blood collection. 
4. Within 30 minutes of the blood draw, centrifuge samples at room temperature (18-25 
oC) for 20 minutes at 1700 RCF (relative centrifugal force.) 
 
Note: Check to see that the tube is in the proper centrifuge adapter. Excessive centrifuge 
speed (over 2000 RCF) may cause tube breakage, exposure to blood and possible injury. 
To calculate the correct centrifuge speed for a given RCF, use the following formula: 
 
 
The variable “r” is the radial distance in centimeters from the centrifuge center post 
to the tube bottom, when the tube is positioned horizontally. RCF is the desired 
relative centrifugal force, 1700 in this case. (2.5cm = 1 inch). 
5. After centrifugation, mononuclear cells and platelets will be in a whitish layer just 
under the plasma layer (see below figure). 

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6. IMMEDIATELY after centrifugation, collect the plasma layer and aliquot into labeled 
screw cap cryovials at 1mL per vial and freeze at -80oC. Plasma samples will be used 
in ELISAs as outlined in section 9.3. 
Collect PBMC according to the following procedures: 
o Gently invert each vacutainer tube 3 times to resuspend leftover layer (which 
contains the PBMCs). Under sterile conditions, decant the liquid from the 
Vacutainer CPT tubes into a 50 ml conical tube with cap. Discard the 
vacutainer containing red blood cells. 
o Centrifuge the conical tube at 300 RCF for 10 minutes at 4oC. 
o Aspirate as much supernatant as possible without disturbing the cell pellets. 
This should be achieved by progressively tilting the tube while having the 
pipette tip touch the side of the tube. Do not lower pipette tip close to the cell 
pellet. 
 
PBMC Counting 
7. Add cold PBS to bring the volume to 10 ml. Re-suspend cell pellets by tapping the 
tube with fingers, then mix by pipetting gently to get an equal cell distribution. 
8. Use either an electronic coulter counter or hemocytometer to determine the number 
of cells/mL.. 
 
PBMC Aliquoting: 
9. Centrifuge the original cell suspension from Step 7 at 300 RCF for 10 min at 4oC. 
10. Immediately after centrifugation, carefully remove and discard supernatant 
without disturbing the cell pellet. 
11. Based on the cell count, add appropriate amount of PBS to adjust the cell 
concentration to 1 x 106/mL. Re-suspend the cell pellets by tapping the tube with your 
finger until no clumps are visible. 
12. Label the appropriate number of microcentrifuge tubes according to the number of 
cells available. 

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13. Aliquot the cells to microcentrifuge tubes: 
• Aliquot cells at 1 mL (1 x 106 cells) per tube. If there are not enough cells for 
two tube, aliquot 1 mL (1 x 106 cells) to one tube, and the rest to the other tube. 
• Centrifuge the tubes at 2862 RCF for 5 minutes at 4oC. 
• Carefully remove all remaining supernatant from the microcentrifuge tube, 
again being careful not to disturb the pellet. 
• Freeze the PBMC pellets immediately at -80oC. 
 
Preparation of Platelet Rich Plasma 
 
1. Collect whole blood in labeled 4.5 mL CTAD blue hemogard-top vacutainer tube 
(BD Medical #367947) tubes and mix thoroughly by inversion. 
2. Centrifuge the blood tube at 180 x g (RCF) for 12 minutes at room temperature. 
*NOTE: The RCF varies according to the centrifuge rotor radius. The formula for computing 
RCF from rotation speed and centrifuge radius is RCF = 11.2r (RPM/1000)2, where r is rotor 
radius, in cm, and RPM is the rotations per minute setting of the centrifuge. 
3. Without disturbing the bottom red blood cell layer, slowly and carefully collect most of 
the platelet rich plasma (PRP) from the top layer of the tube (within 5mm of meniscus) using 
a P- 1000 pipettor. Transfer the PRP into a pre-labeled 15 mL polypropylene tube. Discard 
the remaining red blood cell layer appropriately. 
4. Centrifuge the 15ml tube containing the PRP at 1800 x g (RCF) for 10 minutes at 
room temperature. A white pellet should be visible at the bottom of the centrifuge tube. 
5. Gently remove and discard all of the plasma supernatant using a P-1000 pipettor 
without disrupting the white platelet pellet. 
6. Add 1mL of sterile PBS to the pellet and gently re-suspend the pellet by pipetting up and 
down. Transfer the entire suspended pellet to the previously labeled 1.5ml microfuge tube 
OR cryovial. 
7. Centrifuge the cryovial OR microcentrifuge tube at 450 x g for 5 minutes at 
room temperature. Discard the supernatant. 
8. Record the date and time of collection and the subject‟s initials on the label in permanent 
ink. Secure the label by wrapping with clear tape. 
9. Quick freeze the pellet by embedding the cryovial (or microfuge tube) in dry ice for 
10 minutes and then transfer the frozen cryovial into a secure, monitored -70°C freezer. 
 
Samples will be kept in -80◦C at the following 
address: Meric-Bernstam lab, 
M.D. Anderson Cancer Center 
1515 Holcombe Blvd. Room T5.3849 
Houston, TX 77030 

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APPENDIX I 
IMMUNOHISTOCHEMICAL (IHC) MARKER TEMPLATE FOR 
INTEGRAL MARKERS IN CLINICAL TRIALS 
Immunohistochemical (IHC) Marker Template 
For Integrated Markers in Clinical Trials 
 
 
This is a template to describe the analytical and clinical performance of an assay that is essential 
for performance of a trial. It will be used to assess whether assays are ready for use in a trial by 
Disease Steering Committees and CTEP. The FDA may also use it to evaluate integral assays 
and diagnostics for their pre-IDE evaluation. Not all parameters may be known a priori. Please 
enter as much information as you can and N/A for not available or applicable where appropriate. 
 
 
This template requires detailed information that may be known only by laboratorians, scientists 
who work in clinical laboratories, and should be collaborating closely with clinical trialists. 
Please be sure to collect the appropriate responses before filling out this form. The template has 
the following sections with information needed from trialists and laboratorians:  
 
 
1. Assay, Patient and Specimen Information –Trialists and Laboratorians  
 
2. Primary Antibody Characteristics – Laboratorians  
 
3. Design of Immunohistochemical Assay - Laboratorians  
 
4. Assay Performance – Laboratorians  
 
5. Laboratory Information – Trialists and Laboratorians  
 
 

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Section 1. Assay, Patient and Specimen Information 
 
A. Name of marker (Please use HUGO Gene Nomenclature Committee (HGNC) gene or protein 
name for molecular marker or the Atlas of Genetics and Cytogenetics in Oncology and 
Haematology for cytogenetic or FISH markers) 
HGNC Site: http://www.genenames.org/ 
Atlas Site: http://atlasgeneticsoncology.org/index.html 
 
     Marker name:  pAkt (Ser473), pS6(235/236), pS6(240/244), VEGFR, IGF-1R, 
p4EBP1(Thr70) 
 
B. How will assay and its marker be used in clinical trial? 
     __ Integral Marker     __ Integrated Marker      _X_ Research Marker 
 
• Integral markers are required for the trial to proceed (e.g., patient eligibility, assignment 
to treatment, stratification, risk classifier or medical decision-making -often requires 
performance in a CLIA laboratory).  
• Integrated markers are performed on all or a statistical subset of patients but are not used 
for medical decision-making.  
• Research markers are all other assays and commonly referred to as correlative research.  
• For other definitions, please see References at end of form.  
 
B1. Assay Purpose 
_X_  Treatment Assignment  __ Eligibility Criterion  
__ Stratification Factor  
 
__ Other (Specify) ________ 
 
 
C. Assay type 
_X_ IHC     __ ISH      __ FISH     __ ELISA     __ Microarray     __ RT-PCR   
    __ Other (Specify)  _______ 
 
D. Will the assay be performed in a central reference CLIA lab, multiple CLIA-certified labs, or 
in research labs?  
    __ Central Reference CLIA Lab     __ Multiple CLIA Labs     _X_ Research Labs 
 
 
E. Anatomic source of specimens (organ site) 
_All Comers 
 
E1. Type of Specimen        __ N/A 
__ ascites         __ bone marrow      __ cell       __ normal      __ plasma 
    __ serum 
__ blood           __ buccal mucosa   __ CSF      _X_  tumor       __ skin            __ pleural 
fluid 
__ urine            __ Other (Specify)   ________  
 
 
 
 
E2. Tissue collection    

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__ mandatory (must be performed on trial)   
 
 
_X_  mandatory on consent (must be performed when consent obtained) 
 
__ voluntary 
 
__ not specified 
 
F. Patient conditions or co-morbidities that may affect assay and must be noted: NA 
 
G. Preanalytic Specimen Requirements 
 
G1. Maximum Warm ischemia time (= time from cutting blood supply to removal from 
body) allowed in minutes if known  __ Known       _X_  Unknown 
 
 
Please specify if known    ____ Minutes 
 
 
G2. Maximum Cold ischemia time (= time from removal from body to being frozen or 
put into preservative) allowed in minutes if known   __ Known      _X_ Unknown 
      Please specify if known    _____ Minutes 
 
 
G3. Type of stabilization of Specimen:  _X_  Fixed     __ Frozen     __ Both 
 
 
G3a. If fixed, what fixation buffer?     __ Bouin’s        _X_  10% Neutral Buffered 
formalin 
     __ Other (Specify)  ________ 
 
G3a1. What is the shortest fixation time allowed (Hours or fractions thereof)? 
        _6 hrs_ 
G3a2. What is the longest fixation time allowed (Hours or fractions thereof)? 
        _24 hrs  
 
G3b. If frozen, how will the specimen be frozen? 
      _X_  Flash Frozen (to -80°C) 
 
      __ Embedded in OCT and then frozen 
 
      __ Cryopreserved with controlled rate freezing 
 
   H. How will the specimens be stored? 
    __ -20°C  
 
    __ -80°C  
 
    __ -100°C to -130°C  
 
    __ Vapor Phase Liquid Nitrogen  
    __ 4°C  
    __X_  Room Temperature 
 

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I. Specimen size to be stored     __Inches     __ Centimeters 
 
Length _______ 
 
Width   _______ 
 
Height  _______ 
 
 
J. Tissue section thickness on slide in microns 
     _4  
 
K. Antigen retrieval solution/procedures 
     _Slides will be subjected to HEIR (heat induced epitope retrieval) in 10 mmol/L citrate buffer 
(pH 6.0) for 20 min.   Endegenous peroxidase activity will be blocked for 10 minutes in 0.3% 
hydrogen peroxide.  Slides will be blocked with 1% goat serum for 1 hour at room temperature 
and subsequently primary antibodies will be dispensed on tissue 
    
Section 2. Primary Antibody Characteristics 
 
A. Source of primary antibody (purchased from xxx as lot # xxx, or generated in house, etc.) 
    purchased from Cell Signal 
 
B. What was the immunogen  
    __ Protein     __ Peptide     __ Oligosaccharide     __ Phosphorylated Protein 
    __ Other (describe)  ______ 
 
 
B1. Species of immunogen (e.g., human or mouse gene product)  
  X  Human      __ Mouse      __ Recombinant      __ Other (Specify) ________ 
 
B2. Are there specific isoform(s) of the immunogen that are recognized (e.g., one or all 
isoforms or unknown)? 
  __  One Isoform          __ All Isoforms       X   Unknown 
 
B3. Preparation of immunogen  
  __ Purified Protein      __ Recombinant     __ Synthetic Peptide     __ Oligosaccharide 
 
C. Other attributes of the primary antibody  
 
C1. 
 X Monoclonal      __ Polyclonal 
 
C2. 
     Human  
 
    
__ Mouse  
 
    
X   Rabbit    
   
__ Goat 
 
 
__ Horse 
 
 
__ Chicken 
 
 
__ Other (specify)  ________ 

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D. How was the antibody specificity demonstrated? 
__ IHC      X Western Blot      __ Immunoprecipitation      __ Immunocompetition 
__ Other (specify)  ________ 
  
D1. Are there band(s) at the expected mass(es) on a Western blot?  
 
X Yes     __ No     __Unknown 
 
     If no, please explain  ________ 
 
 
D2. Is immunostaining abolished in knock out/knock-down cells or with epitope-
absorbed antibody? 
 
__ Yes     __ No 
     X Unknown 
 
D3. Is immunostaining abolished when antibody absorbed or blocked with epitope? 
 
__ Yes     __ No 
     X Unknown 
 
E. What is the targeted organ/tissue/cell (e.g., normal melanocytes, breast ductal carcinoma)? 
    all tissue types_ 
 
E1. What non-targeted organ/tissue/cell is also stained? 
      _positive and negative controls_______ 
 
F. Have any cross-reactive proteins or peptides been identified that may confound interpretation 
of IHC? 
 
__ Yes     X No 
     __ Unknown 
 
If yes and known, what are they?  _________ 
 
K. Is the antigen stable when the period between tissue sectioning and staining is 
 
__ <7 days     __ 7-30 days     __ >30     X  Not Known 
 
    
Section 3. Design of Immunohistochemical Assay 
 
A. Assay Design (Complete assay details are needed if multiple labs will perform the assay). 
 
A1. Describe the platform of the assay, e.g. instrument (manufacturer, model, UDI 
number if known) 
 
A1a. Platform ________ 
 
A1b. Manufacturer _ Thermo Scitific Autostainer 

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A1c. Model Number ________ 
 
A1d. UDI Number (Universal Device Number) AS1014A0905 
 
A1e. Is the platform cleared or approved by the FDA 
       __ Yes     __ No     X Unknown 
 
A2. Is there an SOP? 
       __ Yes     __ No     X Unknown 
 
A2a. Is the SOP attached as an Appendix? 
      __ Yes     X No      __ Unknown 
 
B. Type of Immunoassay 
 
B1. Is the assay qualitative, semi-quantitative or quantitative? 
      X Qualitative     __ Semi-quantitative     __ Quantitative 
 
B1a. If an image analyzer is used, what manufacturer and model was used? 
       ________ 
 
B1b. Is it cleared or approved by the FDA? 
       __ Yes     __ No     X Unknown 
 
B2. Nature of the reporter signal 
      ________ 
 
B3. Assay method (e.g. direct, indirect, 3-step immunoperoxidase assay) 
      __ direct     X indirect     __ 3-step Immunoperoxidase      
      __ other (specify) ________ 
  
B3a. What secondary reagent(s) is used for the indirect or 3-step assay 
     _polymer detection kit_______ 
 
C. Are there positive and negative controls for the assay? 
 
X Yes     __ No     __ Unknown 
 
C1. If there are controls, what are they? 
     468 DMSO treated cell lines and ZR75-1 MK2206 treated cell lines 
 
 
D. What is the smallest specimen that can be analyzed by the assay?     <1.0__ cm 
 
D1. Is the minimum specimen size determined by a particular characteristic of the tissue? 
      __Yes     __ No     _ X  Unknown 

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D1a. If so, is it 
       __ Number of cell nuclei     __ Nuclear area  
       __ Cytoplasmic area      _ X _ Other (specify)  _at minimum must have 20% 
tumor____ 
  
 
    
 
Section 4. Assay Performance 
 
A. Details regarding how the analysis is measured 
 
A1. What statistical test(s) were used to validate the assay results 
     _ no statistical test was used.  Pls see section B for reproducibility of assay for 
validation purposes _______ 
 
 
A2. How was a clinically relevant threshold selected? 
      __ Literature     __ Pilot Clinical Study     __ Medical Practice Guidelines 
      __ Non-clinical data (e.g., in vitro or in vivo animal)     _ X _ Other (specify) _______ 
 
A3. Were results obtained on retrospective or prospective data sets?  
      __ Retrospective     _ X _ Prospective     Sample Size __30_____ 
  
 
A3a. Training sets or other validation method 
     X Separate Training & Validation Sets     __ Other Method (specify) _____ 
 
 
A4. What is the cut-off? ________ 
 
A5. How well was the cut-off validated before using it in these trials? ________ 
 
A6. Were assay conditions standardized to minimize variance, (e.g., automated tissue 
processors and/or strainers)?  
      X Yes     __ No     __ Unknown 
 
If yes, what tissue processor/stainer was used? __Thermo Scientific 
Autostainer___ 
 
A7. Were calibrators/controls used? _ X Yes     __ No     __ Unknown 
 
A7a. Were the controls stained as separate slides with slides?  
  
X Yes     __ No 
     __ Unknown  
 
A7b. Were the controls included in each slide and stained as internal controls?  
 __ Yes     __ No 
     __ Unknown 

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A7c. Were the controls not stained in each staining run?  
 __ Yes     __ No 
     __ Unknown 
    
 
B. Reproducibility of assay 
 
B1. Was reproducibility assessed? 
      X Yes     __ No     __ Unknown 
 
If yes, please describe the specimen type(s) used  
   used cell lines of known protein expression confirmed by Western Blot to make 
positive and negative controls. ______ 
 
If no, please explain  
   ________ 
 
B2. How many replicates were done?  _ Antibody was titered prior to running 5 sets of 
10 known positive and negative controls. ____ 
 
B3. What is the intra-lab reproducibility (%CV)?  __95%___ 
 
 
B4. What is the inter-lab reproducibility (same specimens, different lab, and number of 
different technicians)? __95%___ 
 
B4a. How many on the same specimens? 
_50 total of each positive and negative______ 
 
B4b. How many different labs? 
_one______ 
 
B4c. How many different technicians? 
_______ 
 
B4d. What types of specimens (e.g., tissue sections, TMA)? 
       ___cell pellet____ 
 
B4e. Over how many different days? 
       ___several weeks____ 
 
B4f. How many readers? 
       _one____ 
 
B5. What is the agreement between readers?  _____ 
 

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B5a. How are differences resolved? 
      X  Different runs of the same assay 
      __ Different runs of another assay of the same technology 
      __ Different runs of another assay of a different technology 
      __ Different reading by the same reader or instrument 
      __ Different reading by a different reader or instrument 
      __ Panel or arbitration 
      __ Other (please specify) _______ 
    
 
 
C. Image Measurement 
 
C1. What strategy was used to select the fields to be analyzed? 
     ________ 
 
C2. How was a threshold to distinguish positive from negative determined? 
     ________ 
 
C3. How were the cells of interest distinguished from other cells? 
     ________ 
 
C4. Was reference material used to generate a standard curve? 
      __ Yes      __ No      __ Unknown 
 
 
C4a. What was the reference material? 
      ________ 
 
C4b. Has it been cleared by the FDA? 
      __ Yes      __ No      __ Unknown 
 
D. Assay Discrimination 
 
D1. What is the accuracy of the assay for detecting the analyte?  _____ 
 
D2. How are staining and tissue artifacts identified and handled (especially if image 
analysis is used)?  ________ 
 
   
 
Section 5. Laboratory Information 
 
A. Will the assay be performed in a research or clinical lab?  
     X  Research     __ Clinical 
 

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B. Does the lab meet GLP standards 
     _ X  Yes     __ No     __ Unknown 
 
C. What is the training and experience of the Technician/Operators? _ Senior Histotechnologist 
has 20+ years experience in processing IHC assays _______ 
 
 

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