UCLA School of Dentistry, MD Anderson Cancer Center, Fred Hutchinson Cancer Research Center

SSU01 Interim ReportStudy Architecture

2

Samples Available

Current Status Goal

- 133 samples passed QC- ~80 remaining to be assayed

210 Classified Samples- Passed QC- Excluded from Screen

Failures

SSU01 Interim Analysis Workflow

• Test biomarker panel for Sjogren Syndrome– 4 autoantibodies using Lifetech

protoplex panel (no standard curve)

– 3 autoantibodies using in-house developed assay and EMD millipore assay (with standard curve)

• Performance of triplicate tests on 210 blinded Sjogren syndrome patient samples

Key Objectives

• Sensitivity and specificity data on blinded samples

Key end products

Objectives and End Products Desired

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SSU01 INTERIM ANALYSISKey Parties Involved

UCLA

• Project Lead– David Wong dtww@ucla.edu

• Clinical Coordinator– David Akin dakin@dentistry.ucla.edu

• Scientists– Stergios Katsiougiannis stergioskats@ucla.edu– Shigeo Ishikawa shigeo_ishikawa2011@yahoo.co.jp– Michael Tu emtu@ucla.edu’

MDA• Biostatistics

– Ziding Feng ZFeng3@mdanderson.org

FH

• Information Exchange– Litwin, Paul E plitwin@fredhutch.org– Tim M Monahan tmonahan@fhcrc.org

UCLA

MDA

FH

SSU01 Interim Analysis Workflow

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- UCLA finalizes raw data format

UCLA ProtocolDev.

UCLA Finalizes data formats

UCLA Test Run

UCLA – MDA blinding

Process Samples

- UCLA sends MDA complete list of samples

- MDA randomizes blinded samples for testing

- Scheme sent to UCLA and FH

- UCLA will process samples according to protocol. - Raw files and

compiled files sent to FH.

Biostatistics

- FH processes data and sends to MDA

- UCLA finalizes assay development and protocol used for study.

Preparation Interim Analysis

Preparation and Interim Phases

- Data sent to FH in “test run”

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Workflow and Dependencies (If the processes are dependent they are the same color)

Task

• QC of remaining samples (Stergios)

• 3-Plex Assay (Stergios)– Finalization of Protocol for 3-Plex Panel – Finalizing data format– Mock-run of 3-plex using in-house saliva

samples on 78 different saliva samples

• 4-Plex Assay (Stergios)– UCLA performs initial optimization– Finalizing data format for 4-Plex

• Mock Run/ Data handshake with FH

• Data for randomization – Dave Akin sends list of barcodes for samples to

Ziding Feng– Ziding Feng randomizes and sends randomized

wells/plates to UCLA. Barcodes 1-78 will be randomized across plates 1-3, 79-156 across plates 4-6,and 157-210 across plates 7-9

• Validation and analysis– Stergios performs protocol on samples– Michael Tu compiles data file for FH, transmits

compiled data and raws to FH– FH sends data to MDA– MDA Analysis and compilation of results

4-10 11-17 18-24 25-31

January

1-7 8-14 15-21 22-28

Februrary

1-7 8-14 15-21 22-28

March

SSU01 Interim Analysis Workflow

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Data Formats and LabelingFH send sample list and patient records to MDA for randomization (XLSX file)

- QC failures should be excluded- Screen failures should be excluded

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1 2 3 4 5 6

Experimental WorkflowDays of Experiment Work

Read

Read

Read

Read

Read

4 Plex Panel

3 Plex Panel

123

123

456

789

456

Read

789

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Data Formats and LabelingMDA to UCLA XLSX file with barcode IDs and randomization (2 files total, one for 3plex and one for 4plex)Example of the 3plex format:

• Triplicates• 1 replicate/ plate• 78 samples/plate• 9 plates total

3plex (a-enolase, cathespin D, B2M)

1,2,3

4,5,6

7,8,9

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Data Formats and LabelingMDA to UCLA XLSX file with barcode IDs and randomization (2 files total, one for 3plex and one for 4plex)Example of the 4plex format:

• Triplicates• 1 replicate/ plate• 78 samples/plate• 9 plates total

4plex (SSA, SSB, Histones, Transglutaminase

1,2,3

4,5,6

7,8,9

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Data Formats and LabelingUCLA Experimental DataMagpix generated CSV (18 files total, 9 for the 3plex and 9 for the 4plex)

Example for 3plex output

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Data Formats and LabelingUCLA Experimental DataMagpix generated CSV (18 files total, 9 for the 3plex and 9 for the 4plex)

Example for 4plex output

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Data Formats and LabelingRaw Output to Compiled File for UCLA to send to FHOne XLSX file with all the results compiled together (18 files total)

UCLA will compile the raw output data

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Data Formats and LabelingConventions for Naming Files

Title Step in Process Data Files and Naming Convention Links to file

UCLA -> FH XLSX file with list of QC failed samples (1 file total)

FH (to correlate QC’ed sample+Screen Failures to patient records) -> MDA

XLSX file with barcode ID (1 file total)

MDA (randomized list) -> FH -> UCLA

XLSX file with barcode IDS and randomization (2 files total, one for 3 plex and one for 4plex)

3plex well layout link

4plex well layout link

UCLA Experimental Data

Magpix generated CSV (18 files total, 9 for the 3plex and 9 for the 4plex)

Naming convention: [3 or 4 Plex]_[Plate_number]Example: 3Plex_Plate1, 4Plex_Plate8

3plex raw

4plex raw

UCLA (compiled data) -> FH (associates with patients) - > MDA (Analysis)

One XLSX file with all the results compiled together (54 file total) Compiled data

Standard Curve 3plexStandard Curve 4plex

Associates Patient ID’s with patient

data (unblinding)

Summary Overview of Workflow

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UCLAFH MDA

ArrangesPlates

In Random order

UCLA Receives

Design and Performs

Experiments

3-Plex Layout,4-Plex Layout

Experiment performed over 4 days

Performs QC of Samples and informs FH of

failed QC samples

18 Raw Data CSV files

9 File holding 4plex QC (XLS)

18 File Holding Patient Marker Levels (XLS)

9 File holding 3plex QC and Standards (XLS)

UCLA Compiles Data

Analyzes Data

Correlates sample ID’s

to patient records and

removes screen failures

Clinical Collection of Samples

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1. Six Clinical Tests2. Up to 10 weeks

1. Rheumatologist2. Opthalmologist3. Oral Medicine

3. Expenses4. Diagnosis ~6.5 years5. B-cell lymphoma

S

Saliva Biomarker Test

Literature References

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Hu, S, Gao, K, Pollard, R, Arellano-Garcia, M, Zhou, H, Zhang, L, Elashoff, D, Kallenberg, C G, Vissink, A, and Wong, D T Preclinical validation of salivary biomarkers for primary Sjogren's syndrome. Arthritis Care Res (Hoboken), 2010; 62: 1633-1638. PMCID: 2995446.

1) Three salivary protein markers b2M, cathepsin D and a-enolase yielded an ROC of 0.99 in distinguishing pSS from non-pSS subjects

Hu, S, Vissink, A, Arellano, M, Roozendaal, C, Zhou, H, Kallenberg, C G, and Wong, D T Identification of autoantibody biomarkers for primary Sjögren's syndrome using protein microarrays. Proteomics, 2011; 11: 1499-1507. PMCID: NIHMSID # 323544.

2) Four Salivary autoantibody biomarkers, anti-transglutaminase, anti-histone, anti-SSA and anti-SSB yielded an ROC of 0.99 in distinguishing pSS from non-SS subjects

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ReferencesLiterature