Validation of the Archer FusionPlex Solid Tumor Panel in the JAX Cancer Treatment ProfileTM

Samantha Helm1, Aleksandra Ras1, Vanessa Spotlow1, Kevin Kelly1, Susan Mockus1, Cara Statz1, Guruprasad Ananda1, Joan Malcolm1, Gregory J. Tsongalis1, 2

1The Jackson Laboratory for Genomic Medicine, Farmington, CT. 2Dartmouth Hitchcock Medical Center and The Audrey and Theodor Geisel School of Medicine at Dartmouth, Lebanon, NH.

A comprehensive somatic tumor profile with associated treatment selection options requires the detection of gene fusions. After evaluating the analytical validity of multiple methods of gene fusion detection, it was determined that the Archer FusionPlex Solid Tumor Panel (AFPSTP) best compliments the JAX Cancer Treatment ProfileTM (JAX-CTP) clinical test in terms of workflow, specimen requirements and turnaround time. Here we describe our analytical validation process for the AFPSTP assay.

This analysis outlines the clinical validation of the incorporation of AFPSTP into the JAX-CTP test system. Once incorporated, the AFPSTP assay will accomplish the goal of making JAX-CTP a more comprehensive somatic tumor profiling assay without affecting the current acceptable turnaround time or required input material.

JAX® Genomic Medicine | 860-837-2391 | www.jax.org/clinical-genomics | samantha.helm@jax.org

Background

Conclusions

CTP™ Workflow with Fusion Detection Assay Incorporation

Observed False Positives*

Wild-Type Read Through Fusion Events

Homologous Gene Mispriming Fusion events

ADCK4-NUMBL

TEX40-ESRRA

ETV4-ETV1

ETV1-ETV4

PRCKG-PRCKB

*The newest version of the Archer analysis software, released after the completion of the validation, eliminated the presence of mispriming calls and

incorporated a method of identifying wild-type read through transcripts and flagging them as such.

All but one of these fusion events was previously identified. The one novel fusion was confirmed using TaqMan RT-PCR. In addition to the expected fusions, 4 false positive events were detected, 2 due to mispriming and 2 determined to be WT read through transcripts.

Specimen Detected Fusion HorizonDx EML4/ALK Positive EML4 → ALK variant 1

HorizonDx RET Positive CCDC6 → RET

HorizonDx ROS Positive SLC34A2 → ROS1

HorizonDx Triple Positive

EML4 → ALK variant 1

SLC34A2 → ROS1

CCDC6 → RET

A673 Cell Line EWSR1 → FLI1

VCaP Cell Line TMPRSS2 → ERG

KM-12 Cell Line TPM3 → NTRK1

RPMI-2650 Cell Line BRD4 → NUTM1

NCI-H716 Cell Line FGFR2 → COL14A1

OCI-AML2 Cell Line MBNL1 → RAF1

RT-112 Cell Line FGFR3 → TACC3

M0-91 Cell Line ETV6 → NTRK3

REH Cell Line ETV6 → RUNX1

MDA-MB-175-VII Cell Line TENM4 → NRG1

ASPS-1 Cell Line TFE3 → ASPSCR1

ASPSCR1 → TFE3

PDX1 EML4 → ALK 3b

PDX2 SYN2 → PPARG

The fusion detection inter and intra-assay concordance was found to be 100% and the sensitivity was calculated to be 91.67% at a LOD of 5%. The low level of sensitivity calculated for this assay was attributed to repeated freeze-thaws of the starting material causing degradation and therefor diminished ability to call fusions at low levels of detection.

Results Materials and Methods

AFPSTP was validated using 26 samples: 5 JAX Patient Derived Xenograft (PDX) cases, 4 translocation positive controls, 2 FFPE cancer samples, 1 normal tissue sample, and 14 cell lines. Nine of the cell lines were previously identified as positive for fusion transcripts and 3 lacked detectable fusion events. The validation was executed in 5 phases: (1) confirm that AFPSTP was able to detect known fusion or lack of fusion events in characterized specimens; (2) determine inter-assay concordance; (3) determine intra-assay concordance; (4) LOD and (5) sensitivity.

Day 1: All prep

Extraction

RNA Through Fusion Detection

Day 2

• Random Priming • cDNA Synthesis • Pre-Seq QC • End Repair/dATail • Adapter Ligation • First PCR

DNA Through Target Enrichment

• Fragmentation • End Repair/dATail • Adapter Ligation • Pre-capture PCR • Hybridization

• Second PCR • Quantification • 2nM Dilutions • Pooling • MiSeq

Sequencing

• Hyb Wash • Capture PCR • Quantification • 2nM Dilutions • Pooling • NextSeq

Sequencing Day 3 Day 4

Sequencing

Sequencing

Submission to Archer Analysis Virtual Machine

Day 5

Upon completion of analysis, both sets of data are sent simultaneously to curation to generate one cohesive patient report

Fastq Generation and Submission

to Analysis Pipeline

Specimen Acceptance

n=8

n=24 n=8 n=8

0%10%20%30%40%50%60%70%80%90%

100%

2.5% 5.0% 10.0% 15.0%

Sen

siti

vity

% of Fusion Positive Sample in 200ng Input Material

Limit of Detection

Abstract 3630