This poster illustrates the steps taken for clinically validating and incorporating the Archer Fusionplex workflow into an existing clinical assay.

1. 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.
ResultsMaterials 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%
Sensitivity
% of Fusion Positive Sample in 200ng Input Material
Limit of Detection
Abstract 3630