Table 2. OncoNetwork validation of the Ion AmpliSeq™ RNA Fusion Lung

Cancer Research Panel

ALK concordance

• 130 previously characterized FFPE lung tumor samples were sequenced

• 44 were known to contain an ALK fusion based on current detection methods, of those, 40 were

confirmed through AmpliSeq™ and 3 were confirmed via 3'-5' imbalance.

• There were no false positives observed in this data set

• 1 RET and 3 ROS1 fusions were detected from samples not previously tested for RET and ROS1

fusions

• >96% concordance – all non-concordant samples will be retested by FISH

Figure 8. Sensitivity – Ion Reporter™ heatmap of target counts from ALK

positive FFPE lung RNA titrated into fusion negative FFPE lung RNA

• FDA requires FISH assays to have a limit of detection above15% to be positive

• EML4-ALK fusion was detected down to10%

• Similar experiment performed by ARUP – Serial dilution of H2228 (ALK+ cell line) into RNA from

from ALK-negative FFPE tumor sample

• Detected the ALK fusion at 50%, 10%, and 5%

• Detected the ALK fusion in 1 of 2 at 1%

• Experiment also performed by titrating H2228 cell line into Human Brain Reference RNA (negative

for fusions) at 100%, 50%, 15%, 5%, 1%, and 0%

• Detected ALK fusion down to 1%

Figure 7. Ion Reporter™ heatmap of target counts from fusion positive cell

line mixture showing high reproducibility between OncoNetwork sites

Figure 6. ALK 3’-5’ expression imbalance of 75 well characterized FFPE lung

cancer samples

Figure 5. AmpliSeq™ RNA Lung Fusion single sample workflow in Ion

Reporter™ 4.2

Figure 4. Library and sequencing workflow for detection of fusion

transcripts

Figure3. 5’ and 3’ Expression Imbalance Amplicons

Figure 1. The OncoNetwork Consortium

Verification of an Ion AmpliSeq™ RNA Fusion Lung Cancer

Research Panel, workflow, and analysis solution: an

OncoNetwork collaborative research study Jose Cienfuegos1, Kelli Bramlett1, Susan M. Magdaleno1, Angie Cheng1, Rosella Petraroli1, Mary Budagyan1, Fiona Hyland1, Rajesh Gottimukkala1, Orla Sheils2, Bastiaan Tops3, Delphine Le Corre4, Henriette

Kurth5, Helene Blons4, Eliana Amato6, Andrea Mafficini6, Anna Maria Rachiglio7, Anne Reimann8, Christoph Noppen5, Chrysanthi Ainali1, Jin Katayama1, Renato Franco9, Harriet Feilotter10, Paul C. Park10,

Jeoffrey Schageman1, Ian Cree8, Jose Luis Costa11, Alain Rico1, Aldo Scarpa6, Jose Carlos Machado11, Kazuto Nishio12, Nicola Normanno7, Marjolijn Ligtenberg3, Cecily P. Vaughn13, Ludovic Lacroix14, Pierre

Laurent-Puig4. 1Life Technologies, CA;2Trinity College, Dublin, Ireland; 3Radboud University Medical Center, Nijmegen, Netherlands;4Université Paris Descartes, Paris, France; 5VIOLLIER AG Basel,

Switzerland; 6ARC-NET University of Verona, Italy; 7Centro Ricerche Oncologiche Mercogliano, Italy; 8Warwick Medical School, United Kingdom; 9Surgical Pathology, Instituto Nazionale Tumori

"Fondazione Pascale", Napoli, Italy; 10Queen's University, ON, Canada; 11IPATIMUP, University of Porto, Portugal; 12Kinki University Faculty of Medicine Osaka, Japan; 13ARUP Institute for Clinical and

Experimental Pathology, Salt Lake City, UT; 14Institut Gustave Roussy (IGR) Paris, France

ABSTRACT

Fusion transcripts resulting from translocation events in the oncogenic driver

genes ALK, RET, ROS1, and NTRK1 play an important role in lung

adenocarcinoma. There is a need to detect these fusion transcripts with up to

date technologies as they may serve as viable therapeutic targets. We have

utilized a targeted sequencing approach and developed an Ion AmpliSeq™ RNA

Lung Fusion panel, a workflow, and an Ion Reporter™ analysis solution to detect

these known fusion events. The panel detects transcripts from 37 ALK, 9 RET, 15

ROS1, and 11 NTRK fusion variants along with 5 housekeeping genes to serve

as internal controls. The workflow is FFPE compatible requiring an input of only

10 ng of total RNA with the capacity to multiplex up to 16 libraries on a single Ion

318™ chip. The panel was initially validated using 10ng of total RNA from a

cocktail of 3 cell lines containing known lung cancer fusions (H2228 – EML4-ALK

variant 3a and 3b, HCC78 – SLC34A2-ROS1 and LC-2/ad – CCDC6-RET). The

library was sequenced using the Ion PGM™ system and analyzed with the

AmpliSeq™ RNA Lung Fusion workflow in Ion Reporter™. Analysis showed that

the positive control sample contained all expected fusions and control genes and

reported zero false positives fusions. This multiplexed fusion transcript targeted

sequencing solution is currently being validated by all members of the

OncoNetwork Consortium who will test lung cancer tissue samples that have

been well characterized by FISH, real-time PCR, IHC, and/or massarray. Initial

results from OncoNetwork Consortia members reveal 100% concordance

between the AmpliSeq™ RNA Lung Fusion panel and FISH in 25 lung tissue

samples.

INTRODUCTION Life Technologies is collaborating with an international consortium for the

development of a lung fusion panel. The OncoNetwork (see figure below) is

comprised of twelve-translational cancer research institutes with many years of

experience in adopting the latest molecular techniques like next generation

sequencing (NGS)- for lung research. The collaboration resulted in the

development of the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel,

which is currently undergoing validation by all the members. The panel utilizes

the Ion AmpliSeq™ technology for gene fusion detection and targets over 70

fusion transcripts associated with the lung cancer driver genes ALK, RET, ROS1,

and NTRK1. The panel also includes 5’ and 3’ gene expression assays for each

of the four driver genes to use as coarse indicators of a translocation event and

assays for 5 internal control genes. This robust workflow is compatible with

formalin fixed paraffin embedded (FFPE) tissue, requiring only 10ng of total RNA

and able to multiplex up to 16 libraries on a single Ion 318™ chip. An integrated

analysis solution has also been developed to detect present fusions and is

currently available in Ion Reporter™ Software 4.2 as the AmpliSeq™ RNA Lung

Fusion single sample workflow. Currently, validation of the Ion AmpliSeq™ RNA

Fusion Lung Cancer Research Panel and workflow shows high reproducibility

and sensitivity and concordance with other methods of fusion detection.

MATERIALS AND METHODS

OncoNetwork members isolated RNA from FFPE lung tissue using either the

Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit or the Qiagen AllPrep

DNA/RNA FFPE kit. At least 10ng of total RNA was reverse transcribed using the

SuperScript® VILO™ cDNA Synthesis Kit followed by library generation using the

Ion AmpliSeq™ Library Kit 2.0 and the Ion AmpliSeq™ RNA Fusion Lung Cancer

Research Panel. Barcodes were utilized during library generation using the Ion

Xpress™ Barcode Adapters. Libraries were quantified using either the Qubit®

DNA assay or the Ion Library Quantitation Kit then pooled for sequencing. 8-16

libraries were multiplexed and templated using the Ion OneTouch2™ System with

the Ion PGM™ Template OT2 200 Kit. They were then sequenced using the Ion

PGM™ Sequencing 200 v2 kit on an Ion 316™ v2 or 318™ v2 chip. After

sequencing, unaligned bam files were transferred to the Ion Reporter™ Software

4.2 and analyzed using the AmpliSeq™ RNA Lung Fusion single sample

workflow.

RESULTS

Table 1. 3’-5’ expression imbalance empirical cutoff values for ALK, RET,

and ROS1

Fusion Cell line: Dilution series into normal cell line

Library preparation Template preparation

Ion PGM sequencing

Data analysis

Ion Reporter™ Software

Ion PGM™ System

Ion OneTouch 2 System

Entire analytical

workflow contained

within Ion Reporter™

Software v4.2 or later

-0.09-0.075

-0.06-0.045

-0.03-0.015

00.015

0.030.045

0.060.075

0.090.105

0.120.135

0.150.165

0.180.195

0.210.225

0.240.255

0.270.285

0.30.315

0.330.345

0.360.375

0.390.405

0.420.435

0.450.465

0.48

1 3 5 7 9 111315171921232527293133353739414345474951535557596163656769717375

ALK Present

ALK Absent

3’-

5’ I

mbala

nce

Samples with known truths

• The 3’-5’ Imbalance values

were plotted for 75 FFPE

lung cancer samples that

were previously tested with

other technologies.

• There is a correlation

between low imbalance

values that do not contain

a fusion (in red).

• Samples that contain a

fusion (in blue) all contain

higher imbalance values.

• Similar plots were

generated for RET and

ROS1 to provide

guidelines on whether a

translocation took place.

BC

1-1

00

%B

C2

-5

0%

BC

3-

25

%B

C4

-1

5%

BC

5-

10

%B

C6

-5

%B

C7

-1

%B

C8

-0

%B

C9

-10

0%

BC

10

-5

0%

BC

11

-2

5%

BC

12

-1

5%

BC

13

-1

0%

BC

14

-5

%B

C1

5-

1%

BC

16

-0

%

AmpliSeq™

+ -

Current methods + 40 4

- 0 80

Characterized samples AmpliSeq fusion calls

Collaborator

Cell line

Control

ALK

FISH +

ALK

FISH - ROS1+ RET+

Cell line

Control ALK +

Fusion

Neg ROS1+ RET+

INSERM 4 9 18 - - 4 8 19 0 0

Kinky 2 6 6 1 1 2 6 6 1 1

ARUP 1 9 15 3 1 1 8 16 3 1

ARC-NET 2 3 16 - - 2 3 14 2 0

IPATIMUP 1 4 10 - - 1 2 10 1 1

Queens 1 2 15 - - 1 2 15

Total 11 33 80 4 2 11 29 80 7 3

Figure 2. Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel primer

design strategy.

Design Strategy:

• Tm and GC% taken into consideration

• Length of amplicon size should be 115-150bp

• Avoid common SNPs

• Primers to produce amplicons such that

sequenced reads will extend through a fusion

breakpoint by at least 20 nt

• Avoid internal false priming of other amplicons

• Avoid primer dimers

• Avoid gene pair reciprocity (Donor

Acceptor interactions)

• In normal samples, we expect 5’ expression

and 3’ expression to be at equivalent level.

• In samples positive for fusion involving the

driver gene, there will be a significant

imbalance between 5’ expression and 3’

expression

ALK 3’-5’ Imbalance defined as:

[(3’ ALK count -5’ ALK count) /Sum of Expression Control Genes counts] CONCLUSIONS

We have developed an NGS workflow solution that enables simultaneous

detection of multiple variants of ALK, RET, ROS1, and NTRK1 gene fusion

transcripts in a single panel along with an integrated analysis solution.

The Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel shows: • Robust performance starting from low total RNA (10 ng) isolated from formalin

fixed paraffin embedded tissue • Excellent reproducibility : 100% reproducibility on the cell line control results • High sensitivity

• fusion transcripts can be detected in 10% fusion positive tumor RNA in the presence of 90% normal FFPE RNA

• Fusion transcripts can be detected in 1% of fusion positive cell line in the presence of 99% normal RNA

• Good concordance with other methods: > 96% concordance. • Straightforward analysis – complete solution using IR v4.2 software

AR

C-N

ET-1

AR

C-N

ET-2

AR

UP

INSE

RM

-1

INSE

RM

-2

INSE

RM

-3

INSE

RM

-4

Kin

ki-1

Kin

ki-2

Vio

llier

-1

Vio

llier

-2

• A mixture of 3 fusion positive cell

lines (H2228, HCC78, LC-2/ad) was

sequenced 11 times in 5 separate

sites.

• All samples detected the correct ALK,

RET, and ROS1 fusions.

• Each sample showed a consistent 3’

overrepresentation for ALK, RET, and

ROS1.

• The 5 expression control genes were

detected and consistent amongst all

samples.

• Similar results for all other sites

tested (data not shown).

Fu

sio

ns

3’-5

’

Imb

ala

nce E

xp

ressio

n

Co

ntro

ls

For Research Use Only. Not for use in Diagnostic Procedures.

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Fisher Scientific and its subsidiaries unless otherwise specified.