JACOB Central Laboratory Procedures and Sample Management

Where tumor tissue and biosamples are sent To Targos Tumor tissue for HER2 testing to ascertain subject eligibility for study (Ambient) To Covance All ‘on study’ mandatory and optional samples (Frozen)

First Communication with Sites (Except Japan) Confirmation of site’s contact details Correct mistakes and enter any missing information Fax the completed form back within 24 hours Dry ice provider and courier information in Europe Contact Covance CLS with changes

Initial Supplies Review packing list within initial shipment Visit-specific collection kits Visit Cycle 1 Day 1, Cycle 2 Day 1 RCR Tumor Tissue Kit All Sites 5 kits each 3 kits 10 kits To obtain highest quality specimens possible, store collection supplies at recommended 4ᵒC to 25ᵒC (39ᵒF to 77ᵒF)

Initial Supplies Covance CLS Investigator Manual Provided in multiple languages Requisition forms, Licenses, CVs, and Reference Ranges remain in English In China, Requisitions are provided in Mandarin Collection and packaging instructions Laboratory certifications Covance CLS contact information Courier contact information Provided by fax in Europe Please review manual prior to first patient visit

Initial Supplies Quick Reference Guide Laminated Synopsis Provided in most local languages Kit information tips Requisition completion tips Resupply Inventory control tips Laminated Synopsis Collection instructions per visit Packaging Material resupply Covance CLS contact information

Initial Supplies Shipping supplies Bulk supplies Shipping boxes (except Japan) Site specific shipping documents Air waybills, Commercial Invoices, Customs Declaration template, return address labels (except China and Japan) Shipping supplies are provided by courier or Covance CLS, depending on courier arrangements Refer to Covance CLS Investigator Manual and courier arrangements fax for country specific information Bulk supplies Positively charged slides

Ambient Specimens/Tumor Tissue Kit supplied by Covance and shipped to Targos (Germany) Tumor Blocks Tumor Slides (within 48 hours of sectioning)

Covance Initial Supplies: Tissue Collection Review packing list within initial shipment HER2 screening collection kit: Tumor Tissue Kit All Sites 10 kits To obtain highest quality specimens possible, store collection supplies at recommended 4ᵒC to 25ᵒC (39ᵒF to 77ᵒF)

Tissue Collection: Key Points (1/4) Archival Tumor Specimens Study sites should send patients’ tumor specimens to Targos as early as possible during screening period to avoid delay Specimens must be FFPE tumor blocks (10% neutral buffered formalin) If FFPE block (or partial block) unavailable because of country or site regulations, then slides may be accepted Slides should only be submitted if no possibility of submitting blocks Minimum 15 unstained slides requested for central review of HER2 status, MET expression, and exploratory biomarker research

Tissue Collection: Key Points (2/4) Archival Tumor Specimens Failure to follow these instructions could delay results reporting: Fill out requisition form provided in tissue kit De-identify associated pathology report Black-out patient identifiers and replace with subject screening number Ensure ID on path report and block match Insert white copy of completed requisition form and copy of de-identified pathology report in outer pocket of specimen biohazard bag Retain pink copy of requisition form and copy of pathology report in study binder Write screening ID on specimen label on sample container Ship samples at ambient temperature to Targos HER2 testing results will be faxed to you within 3 working days of sample receipt at Targos (for samples delivered before noon)

Example of result report form

Tissue Collection: Key Points (3/4) Archival Tumor Block Ensure block is labelled with block ID If block IDs not used, please record patient screening number on block Place block in plastic bubble wrap padding cut to size Place wrapped block in 60 mL plastic orange top container Place container inside largest compartment of specimen biohazard bag and seal Place specimen biohazard bag into ambient kit box and close Block will be returned to site address on file unless you specify otherwise on Requisition form

Tissue Collection: Key Points (4/4) Archival Tumor UNSTAINED Slides Label slides in a solvent resistant manner (e.g. pencil or diamond etching) with study number BO25114, patient screening number and block ID (do not use adhesive labels) Record date slides were sectioned on requisition form Section the paraffin block as follows: Sections should be 4 microns thick Mount 15 serially-cut (consecutive) sections onto the SuperFrost charged slides provided in the tissue kit SAMPLES SUBMITTED ON INCORRECT SLIDE TYPE WILL NOT BE TESTED!!! Allow slides to dry completely Place slides in slide container provided in tissue kit Wrap slide container in bubble wrap and place inside the specimen biohazard bag and seal Place specimen biohazard bag into ambient kit box and ship slides within 48 hours of sectioning

Tumor Tissue: Requisition Completion Samples are submitted to Targos Full page example

Covance: Kit Inventory Review Kits are automatically resupplied, except: Tissue kits Lost, damaged or expired kits Latin America, Russia, Thailand Review for expired kits in preparation of patient visits Use shorter expiration date first Destroy expired kits or kits/supplies in excess at the end of study Do not return to Covance CLS Inform Covance CLS of lost, damaged, discarded or expired kits

Covance CLS Resupply Requests (1/2) Request following supplies from Covance CLS Replacement of damaged, lost or expired kits All kits in Latin America, Russia, Thailand Shipping supplies (except when provided by courier) Tissue kits Order supplies online: www.covance.com/kitordering Secure website Confirmatory e-mail sent providing work order reference number

Covance CLS Resupply Requests (2/2) Allow minimum of 7-9 working days for the delivery of your resupply order Exceptions: 12 in China 24 in Japan + additional days for customs clearance to receive your resupply order 18

Initial Supplies: Blood sample collections Review packing list within initial shipment Visit-specific collection kits Visit Cycle 1 Day 1, Cycle 2 Day 1 RCR* All Sites 5 kits each 3 kits * Optional RCR consent required for collection To obtain highest quality specimens possible, store collection supplies at recommended 4ᵒC to 25ᵒC (39ᵒF to 77ᵒF)

Blood Sample Collection Kit Content All collection items for specified visit Tubes pre-labelled with test names Write Screening/Subject Number on tube labels 2 extra bar coded labels If tube replacement required 1 2 3 4 5 Serum PCR Serum PCR 1 2 3 4 5

Accession Number A unique identifier assigned to each kit Do not interchange kits between sites or protocols! Do not interchange tubes between kits!

Laboratory Requisitions Completion is mandatory for sample processing Provided in English (except China) All information completed in English Complete all required information and answer questions Collection instructions and shipping condition Ship white copies to Covance CLS with samples Retain pink copies for your records

Requisition Completion Completion is mandatory for sample processing METGASTRIC SHOW LUNCH TO END 23-Apr-17 Requisition Completion Completion is mandatory for sample processing Subject Number 1 To identify your patient Birthdate Complete month field in English (Example: 02 JUN 1968) Day Month Year 1 J U L 1 9 8 Male Female Sex X Collection Date Complete month field in English (Example: 01 JAN 2001) Day Month Year 1 J A N 2 1 3 To calculate specimen stability 24 Hours Clock Collection Time (Record Midnight as 23:59) 08 : 00 Requisition Completed by Full name in capital letters DR JOHN SMITH To assist in site communication Samples Collected by Full name in capital letters DR JOHN SMITH Phone Number +44 0131 732 787 Clear + Consistent + Complete = Query-Free Requisitions

Requisition Completion Provide information using check boxes or written entries Provide collection date (DD-MMM-YYYY) and collection time (24 hr) for each time point Respond  Sample not collected if the sample was not collected Incomplete information triggers queries

Frozen Specimens sent to Covance CLS Frozen (ship day of collection or next day) Serum SM1 Trastuzumab PK Pre-Dose SM2 Trastuzumab PK Post-Dose SM3 Pertuzumab PK Pre-Dose SM4 Pertuzumab PK Post-Dose SM5 ATA Pertuzumab SM6 Serum RCR SM9 HER2 ECD/Ligands Plasma SM7 Plasma RCR Whole Blood SM8 Whole Blood RCR SM = Specimen Management Covance CLS forward samples to specified laboratory for analysis as directed by sponsor Testing not performed and not reported by Covance CLS

Sample Collection Key Points Follow detailed collection instructions provided In Covance CLS Investigator Manual On requisitions within the kits On Laminated Synopsis Ship samples day of collection or next day Maintain at frozen until shipment Good Quality DATA Begins with Good Quality Samples

Sample Collection Key Points Before sample collection Serum and plasma processing Check tube’s expiry date Centrifuge 15 minutes, 1500 – 2000 x g, 4ᵒC, within 60 minutes of blood collection (same conditions apply if no cooled centrifuge available) Do not use expired tubes Ensure you chose correct tubes labelled with correct sample name, patient and sample number Transfer to corresponding transfer tube and invert to mix serum/plasma Sample collection Draw blood sample and invert 8-10 times Aliquot into one tube to obtain ~ 3 mL Clot serum sample at room temperature for at least 30 minutes or until clotted Aliquot within 20 min after centrifugation is finished

Sample Collection Key Points Prepare specimens for shipment Freeze preferably at -70ᵒC If not possible, at -20ᵒC Record sample processing, storage and shipping in Sample log Discard used blood collection and transfer tubes according to local regulations Ship to Covance as soon as possible after collection (either same day or next morning) Special Instructions RCR samples can only be taken if separate RCR informed consent has been given

Sample Collection Key Points Sample tubes vary per specimen type Collect Invert Clot Serum only Centrifuge Except whole blood Aliquot Except whole blood Freeze 6 mL blood sample 8-10 times Clot sample at room temperature for at least 30 minutes or until dotted Serum only 15 minutes, 1500 – 2000 x g, 4ᵒC, within 60 minutes of blood collection (same conditions apply if no cooled centrifuge available) Except whole blood Transfer to 5 mL transfer tube and invert to homogenize the plasma. Then aliquot into one tube to obtain ~1.5 mL. Aliquot within 20 min after centrifugation is finished Preferably at -70ᵒC, if not possible: at -20ᵒC = 15-25ᵒC = 1 inversion

General Packaging Reminders Store samples at appropriate temperature Ambient Frozen -70ᵒC, -20ᵒC Use Covance CLS/courier provided boxes (except Japan) In Japan, no shipping boxes are used Plan ahead for pickup and dry ice requests May require 48 hour advance notice Cancelling your request Consider holiday shipping schedules Consolidate according to shipping temperature One accession number per Specimen Collection Bag and per shipping temperature Complete and send required shipping documents Avoid delays

Shipping Documents (Except Japan) Pre-Printed provided by Covance CLS or courier Air waybills, Commercial Invoices and Customs Declaration template Complete required information Send/attach to appropriate shipping box Ensure Commercial Invoice/Customs Declaration is included Customs Declaration Commercial Invoice

Examples of Queries 1 1 Subject Number Missing, incomplete or inconsistent data on requisitions 1 1 Subject Number Incorrect identifiers on tubes Pat ID____ HER2 ECD 11 K-M Multiple accessions used for 1 visit

Specimen Pending Arrival Report Faxed to sites once per month List samples that are: > 30 days old Any frozen samples cancelled as not received since last report Samples are considered lost when 62 days have passed Reminds sites to send samples to Covance CLS

Synergy with MetGastric study JACOB study accepts HER2 test result from Targos and vice versa In those MetGastric and JACOB overlapping sites (25%), screen for MetGastric study first Where patient screened in MetGastric study has HER2 positive status: Obtain Informed Consent for JACOB Investigator/study nurse registers MetGastric study patient screen number in the JACOB’s IXRS screening module Send results transfer form with detailed patient information to Targos → Tissue sample will be transferred within Targos If site is not a JACOB/MetGastric overlapping site, a referral network is recommended

Jacob/MetGastric Sample Sharing ICF signed for MetGastric Screening Call in IXRS for MetGastric Dispatch FFPE tumor block to Targos (or at least 15 slides) HER2 testing performed at Targos Continue with remaining screening activities until HER2 result from Targos is available HER2- Randomization call in IXRS HER2+ Screen Failed Call in IXRS for MetGastric ICF signed for JACOB Screening for JACOB in IXRS; enter MetGastric screening number as reference Results Transfer form completed by site and sent to Targos Randomization for JACOB in IXRS

Recruitment Challenge: High Screen Failure Rate It is assumed that screening ineligibility rate may be ~85% Primarily driven by requirement for HER2 positivity ~ 22% of patients with advanced gastric cancer are HER2+ But… it’s not impossible: ToGA trial recruited successfully with a ~80% screen failure rate Improve overall screening rates by implementing process of pre-identifying and pre-screening patients This will help reduce screening failure rate not related to HER2 positivity If local pre-screening HER2 result is borderline or equivocal, please still send for central HER2 testing Reviewing pre-screening information will help identify why potentially eligible patients are not considered for screening