References

Evaluation of RNA Purification Protocols for Targeted Deep Sequencing of RNA from FFPE TumorsGeoff Otto1, Kristina Brennan1, Jie He1, Geneva Young1, Doug Horejsh2, Doron Lipson1, Jeff S Ross1,3, Phil Stephens1, Alex Parker1

1Foundation Medicine Inc., Cambridge, MA (USA); 2Promega Corporation, Madison WI; 3Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, NY (USA)

Abstract

Background

Results

FFPE RNA is highly degradeda and can co-purify with inhibitors of downstream enzymatic reactions making comprehensive molecular profiling difficult.

a) von Ahlfen S, et al, 2007. Determinants of RNA quality from FFPE samples. PLoS ONE 2(12): e1261.

b) Lipson D, et al, 2012. Identification of recurrent KIF5B-RET gene fusions from lung cancer biopsies. Nature Medicine

18(3): 382-384.

c) Levin J, et al, 2009. Targeted next-generation sequencing of a cancer transcriptome enhances detection of sequence

variants and novel fusion transcripts. Genome Biology 10(10): R115.

d) Peled N, et al, 2012. Next generation sequencing identifies and immunohistochemistry confirms a novel crizotinib

sensitive ALK rearrangement in a patient with metastatic non-small cell lung cancer. J. Thoracic Oncology 7(9): e14-e16.

A single assay capable of characterizing both the genome and the transcriptome of diverse tumor types using targeted, deep sequencing will improve the application of personalized medicine to cancer. Integrating RNA-seq into our clinical FFPE DNA-seq test, which is highly sensitive to many types of genomic alterations in over 200 cancer related genes, will enable a comparison of changes in gene copy number with digital gene expression levels, facilitate validation of both recurrent and novel gene rearrangements and reveal changes exclusive to the transcriptome such as alternative splicing. Tumor samples are commonly stored as formalin fixed paraffin embedded (FFPE) blocks which degrades nucleic acids and requires careful processing to extract sufficient quantity and quality of DNA and RNA to enable comprehensive molecular profiling. Quantitative recovery of high purity RNA from FFPE tissues is essential to implementation of a targeted RNA-seq cancer assay.

2000

18S28S

Fresh Frozen RNA

FFPE RNA

200Length (nt)

Five FFPE RNA extraction kits were evaluated for RNA yield, quality and laboratory workflow using sets of 8 tumor:normal pairs from four different tumor types. FFPE tissue digestion and RNA purification was performed using a prototype kit for the Promega Maxwell® and commercially available kits from Roche, Agencourt, Qiagen and Omega Bio-tek. The two best performing kits were further compared using targeted, deep sequencing on an Illumina HiSeq™ 2000.

Background: Challenges Working with FFPE RNA

Materials & Methods: RNA-seq Sample Preparation Workflow

10 microns of FFPE tissue

Column-based Purification Bead-based Purification

Omega Bio-tek E.Z.N.A.® FFPE

RNA (OBT)

Qiagen RNeasy FFPE RNA

(Qia)

Roche High Pure RNA Paraffin

(RHP)

Agencourt® FormaPure

(AFP)

Promega Maxwell® RNA

FFPE (PM)

Quantity & Purity: Ribogreen & NanodropQuality: Conversion efficiency of RNA to cDNA

Adaptor ligated libraries created with top 2 extraction candidates

Solution hybrid selectionc

49 x 49 paired-end reads were analyzed using a custom pipeline for detecting changes in gene expression and genomic rearrangementsb

RNA Extraction:

Quality Control:

Library Construction:

Hybrid Selection:

Sequencing & Analysis:

FFPE Input: Summary

References

0 0.05 0.1 0.15 0.2 0.25 0.3

Promega MaxwellRoche High Pure

Tissue Volume (mm3)

0

5

10

15

20

RN

A Y

ield

(ng

/ µL

)

Target Input forReverse Transcription

Hematoxylin and eosin stained slide of a 3 mm Tissue Microarray (TMA).

0.04 mm3

0.14 mm3

0.28 mm3

Intact ribosomal RNA Bioanalyzer RNA Pico analysis of a typical FFPE RNA and fresh frozen RNA.

AUG

EML4 (chr2)

1 11 13

genomicshards

ALK (chr2)

20 23 25 27 29

AUG

EML4 ALK

13 20

Transcription & Splicing

RNA compatible with targeted deep sequencing was successfully extracted from FFPE tissue sections with both the Roche High Pure Paraffin and Promega Maxwell kits.

Targeted RNA-seq on FFPE tissue confirmed a novel KIF5B-RET gene fusion observedby DNA-seq and revealed increased expression of the tyrosine kinase domain of RET.

Targeted RNA-seq detected a canonical EML4-ALK gene fusion that was not detected by the standard of care ALK Break-Apart FISH assay.

A.

B.

C.

D. D. ALK immunohistochemistry confirmed the increased expression of the ALK kinase domain detected by targeted RNA-seq.

C. Targeted RNA-seq detected a canonical gene fusion between exon 13 of EML4 and exon 20 of ALK by sequencing reads spanning the EML4-ALK junction and by increased expression of the ALK kinase domain.

B. Targeted DNA-seq detected novel inter- and intra-chromosomal chimeric reads between ALK intron 19 on chromosome 2, a canonical breakpoint, and 4 different regions of the genome distal to EML4.

A. FFPE section from a 43 year-old never smoker was classified as EML4-ALK fusion negative based upon the results of a fluorescent ALK Break-Apart FISH assay (Abbott Molecular). The results of the fluorescent hybridization test displayed an atypical pattern of double 3' ALK signals (in red) fused with the 5' ALK (in green).

RNA yield vs. total tissue volume from pools of 1 to 8 3 mm TMA cores combined in a single extraction.

The PM kit yielded more FFPE RNA than the RHP kit from small tissue inputs and the PM kit extracted sufficient RNA for molecular profiling from as little as 0.18 mm3.

From the results of testing kit performance on 16 FFPE samples, RHP had the highest yield and good purity and PM had the easiest workflow and acceptable yield and purity.

Position on RET Transcript (bp)

Position on ALK Transcript (bp)1,000 2,000 3,000 4,000 5,000 6,000

ALK

Cov

erag

e D

epth

100200300400500600700800900 Kinase Domain

Targeted RNA-seq confirmed a novel gene fusion, KIF5B-RET, in a NSCLC FFPE tumor sample that was initially detected by DNA-seq and revealed a 30-fold over-expression of the tyrosine kinase domain of RETb.

8642-4-6-8

6

4

2

-4

-6

KIF5B-RETwild-type RET

RET exons 1-11

RET

exon

s 12

-20

0 500 1000 1500 2000 2500 3000 3500

RET

Cov

erag

e D

epth

0

500

1000

1500

2000

2500

3000

3500

4000KIF5B-RETwild-type RET

Cadherin Tyrosine Kinase

JunctionExons 11-12

Log-ratio expression of exons 1-11 and 12-20 across 49 NSCLC samples.

Pure RNA = 2.0

1.9

2.0

2.02

2.03

1.79

39.9

25.6

52.1

40.3

21.6

0 20 40 60 1.8 1.9 2.0 2.1 0 1 2 3 4

Average RNA Yield (ng / µL)Average Purity

(Ratio of 260/280)Processing Time (h)

PromegaMaxwell

AgencourtFormaPure

RocheHigh Pure

QiagenRNeasy

Omega Bio-tekEZNA

Targeted RNA-seq revealed that a complex genomic rearrangement involving EML4 and ALK, fusion negative by ALK Break-Apart FISH, expressed an EML4-ALK fusion transcriptd.

E. E. Pelvic positron emission tomographic computed tomographic scans before and after 4 months of crizotinib therapy showed shrinkage of the primary tumor.

Pre-Treatment 4 Months Post-Treatment