RNA Extraction from Formalin Fixed Paraffin Embedded (FFPE) Tissue Enabling Next Generation Detection of Gene Fusions

Transcription

1 RNA Extraction from Formalin Fixed Paraffin Embedded (FFPE) Tissue Enabling Next Generation Detection of Gene Fusions William M. Rehrauer, PhD Associate Professor University of Wisconsin School of Medicine and Public Health Director of Molecular Diagnostics University of Wisconsin Hospital and Clinics

2 Disclosure(s) William M. Rehrauer no relevant financial relationships with either Promega Corporation or ArcherDX, Inc. or any commercial interests in any of the instruments/products discussed in today s presentation Promega and ArcherDX have provided reagents and technical support for this project. Promega has also supported my attendance at the 2016 AMP meeting Rehrauer/UW Health Molecular Diagnostics are members of the Roche MCOE Advisory Group

3 Precision Medicine in Oncology Molecular Interpretation FDA Approved Off Label Use Clinical Trials Standard of Care Ongoing Reassessment Adapted from Levi A. Garraway et al. JCO 2013;31: Many Advances, Changes and Challenges?

4 Precision Medicine in Oncology Challenge 1 Breadth of: Disease Genes Mutations Cancer Genome Landscapes Vogelstein et al. Science 2013; 339:

5 Precision Medicine in Oncology Challenge 2: Many Types of Molecular Alterations Cancer Genome Landscapes Vogelstein et al. Science 2013; 339:

6 Precision Medicine in Oncology Challenge 3: Many Technologies, Tissue Quantity EGFR exon 19 EGFR exon 20 ERBB2 exon 20 Dideoxy Terminator (Sanger) Sequencing EGFR exon 21 Lung Cancer EGFR T790M BRAF COLD Real Time PCR Melting Curve (FRET) Pyrosequencing MALDI TOF Mass Array KRAS KRAS c.34g>t ERBB2 ALK MET Fluorescence in situ Hybridization ROS Immunohistochemistry

7 Precision Medicine in Oncology Challenge 4: Tissue Quality/Heterogeneity Degraded or poor quality NA Consider alternate sample types Increase functionality of extracted NA Evaluate/implement robust techniques Mutations at low allele fraction Macrodissection Tumor heterogeneity Contamination Aneuploidy (quantitative testing) N T

8 Challenges Extracting Nucleic Acids from FFPE Tissue Formalin fixation crosslinks proteins and nucleic acids Causes degradation/fragmentation of nucleic acids Degree of degradation/fragmentation varies depending on sample type, sample age, and fixation conditions Formalin fixation affects downstream applications Crosslinked nucleic acids are inaccessible to amplification enzymes Formalin residuals inhibit enzyme activity during extraction and PCR Degradation/fragmentation inhibits amplification De-paraffinization often involves xylene or other organics Hazardous to end user Requires special disposal Must be removed during sample pre-processing

9 Precision Medicine in Oncology MET ROS1 RET Lung Cancer PIK3CA Other KRAS EGFR ERBB2 EGFR-mutant cancer: erlotinib, gefinitib ALK BRAF HER2 Mutated NSCLC (Trastuzumab) Dabrafenib/ Trametinib Resistant to inhibitors targeting signaling through EGFR Lynch et al., NEJM, 2004 Paez et al., Science, 2004 Pao et al., PNAS, 2004 BRAFV600E-mutant NSCLC N Engl J Med 2006; 354: June 15, 2006 Combined BRAF and MEK inhibition in BRAF-mutant NSCLC The Lancet Oncology, Volume 17, Issue 7, July 2016,

10 Chemotherapies, Immunotherapy and Targeted Therapies Management of NSCLC Thomas, A. et al. (2015) Refining the treatment of NSCLC according to histological and molecular subtypes Nat. Rev. Clin. Oncol. doi: /nrclinonc

11 Targeting Oncogenic Alterations (NSCLC) Thomas, A. et al. (2015) Refining the treatment of NSCLC according to histological and molecular subtypes Nat. Rev. Clin. Oncol. doi: /nrclinonc

12

13 Precision Medicine in Oncology Breadth of genes, mutations, types of alterations Explosion of targeted therapies Efficiency/Evolution of Testing methods Sample Utilization Expansion of Clinical Trials (options clinicians/patients)

14 UWHC Oncology Testing in Lung Cancer (NSCLC) Ion AmpliSeq Cancer Hotspot Panel v2 (Next Generation DNA Sequencing) bench top footprint semi-conductor chip detection (can be scaled) 2-3 day procedure (sample to DNA variants) genetic variant caller included 50 genes; 207 amplicons; >2,800 mutations genes sequenced only in targeted regions of exons cannot differentiate somatic vs. germline data analysis: diagnosis, prognosis, optimal therapy

15 Promega Maxwell CSC and DNA FFPE Kit Promega Maxwell CSC 1 Maxwell CSC DNA FFPE Kit 1,2 1 For In Vitro Diagnostic Use 2 Intended for use with FFPE tissues collected from human breast, lung or colon Graphical user interface via integrated tablet touchscreen Self Test documented: mechanical operation, heater works, database functional Complete technical manual for Maxwell CSC and protocols on tablet User documentation (network administrator); documents on report (audit trail) Use of UV light can be controlled/documented by administrator Add and update protocols; protocols are selected via barcode Sample tracking capabilities via barcode scanner (50 characters) Reagent kit lot number and expiration date tracking via barcode scanner Software requires user acknowledgement (3 setup steps) Reporting: electronic (USB) or hardcopy via printer

16 Promega Maxwell CSC and DNA FFPE Kit Promega Maxwell CSC 1 Maxwell CSC DNA FFPE Kit 1,2 1 For In Vitro Diagnostic Use 2 Intended for use with FFPE tissues collected from human breast, lung or colon Sample Input Maxwell CSC DNA FFPE kit: 1 x 5μM tissue section (macrodissection) Deparaffinization No organic reagents required for deparaffinization (mineral oil) Lysis Buffer addition/proteinase K Treatment Centrifugation to separate aqueous/non-aqueous layers Decrosslinking 56 C for 30 minute and 80 C incubation for 4 hours (efficient de-crosslinking) RNase treatment of aqueous layer All reagents/disposables are included (no aliquoting/preparation needed) Add to Maxwell cartridge Throughput: 16 samples in <60 minutes hands on time; Maxwell CSC walk away (run time ~30-40 minutes)

17 Benefits of Maxwell CSC FFPE DNA Protocol Safety: benefits of removal of paraffin by mineral oil Efficiency: no repeated deparaffinization/etoh/water steps DNA yield: on average provides an overall greater DNA yield DNA performance: greater functionality (amplification(s), library(s)) Conservation of tissue: reduced our FFPE/slide needs (40-60%) Samples other than FFPE: fine needle aspirations, cell blocks (Cytology) Analyte Analytical Sensitivity % Tumor Sensitivity > %Tumor Sensitivity < %Tumor Sensitivity KRAS Testing 10% 20% 5 x 5uM tissue curls in tube BRAF Testing 5% 10% 5 x 5uM tissue curls in tube EGFR Testing 15% 30% 5 x 5uM tissue curls in tube Cancer Gene Mutation Panel 5% (Coverage) Macrodissection of Tumor 5 x 5uM slides (*3 rd H&E stained) 5 x 5uM slides (*3 rd H&E stained) 5 x 5uM slides (*3 rd H&E stained) 3 x 5uM slides (*2nd H&E stained)

18 UWHC Testing in Lung Adenocarcinoma Mutations EGFR KRAS BRAF ERBB2 PIK3CA MET ROS1 RET PIK3CA Other 1)Promega Maxwell CSC 1 2)Maxwell CSC DNA FFPE Kit 1,2 3) ThermoFisher AmpliSeq v ) ThermoFisher PGM 3 KRAS EGFR ERBB2 ALK BRAF Fusions ALK RET ROS1 other 1)Promega Maxwell CSC 1 2)Maxwell CSC RNA FFPE Kit 1,2 3) Archer FusionPlex System 3 4) ThermoFisher PGM 3 1 For In Vitro Diagnostic use. 2 Intended for use with FFPE tissues collected from human breast, lung or colon. 3 For research use only. Not for use in diagnostic procedures.

19 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC 1 Maxwell CSC RNA FFPE Kit 1,2 TNA 1 For In vitro Diagnostic Use 2 Intended for use with FFPE tissues collected from human breast, lung or colon 3 The studies shown in this presentation are research studies and are not intended for diagnostic procedures ~2 Hours Preprocessing + 45 Minute Instrument Run (~30 min Hands On Time)

20 Fusions SNV/InDel Splicing Expression CTL FusionPlex RNA Comprehensive Thyroid and Lung Panel Parallel, Lyophilized Workflows All possible fusion partners can be detected Expression imbalance verification of fusions AKT1 ALK AXL BRAF CALCA CCND1 CTNNB1 DDR2 EGFR EIF1AX ERBB2 FGFR1 FGFR2 FGFR3 GNAS HRAS IDH1 IDH2 KIT KRAS KRT20 KRT7 MAP2K1 MDM2 MET NRAS NRG1 NTRK1 NTRK2 NTRK3 PDGFRA PIK3CA PPARG PTEN PTH RAF1 RET ROS1 STK11 SLC5A5 TERT THADA TP53 TSHR TTF1

21 Detect gene fusions with the Archer FusionPlex System Archer Anchored Multiplex PCR (AMP ) Chemistry Workflow Total Nucleic Acid or RNA QC: qpcr assay RNA functionality Ion A P1 QC NGS: 1) Unique GSP2 Start Sites 2) If Fusion + Unique Start Sites 3) If Fusion + Spanning Reads

22 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit Quality Control Results TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA

23 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA Quality Control Results N=56 Archer RNA PreSeq (Cp) Specificity (median) Sensitivity (median) ROC "best" cutoff Archer RNA PreSeq (Cp) Specificity (median) Sensitivity (median) % CI (2000 stratified bootstrap replicates)

24 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA Quality Control Results

25 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA 3 56 Samples FFPE-CB FFPE-Tissue FFPE-Tissue DQ-FNA Controls

26 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA 10 Thyroid 3 3 FFPE Tissue FNA-DQ 2 Duplicates 2 Fail 41 Lung FFPE FFPE Tissue FFPE-CB Duplicates Failed

27 Ave Unique GSP2 Control Start Sites Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA 10 Thyroid 2 Duplicates 5 Fail # Slides Extracted % Correlation with ArcherDx 38 2 MET fusions CCDC6 Ret fusion 2 BRAF V600E (TPM3-NTRK1 fusion)

28 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA Lung Duplicates 7 Fail

29 Ave Unique RNA GSP2 Control Start Sites Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit 41 Lung FFPE Samples TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA Duplicates to Assess Tissue 40 Requirements FFPE Tissue FFPE-CB Duplicates Failed Failed even with 4 slides Failed with <4 slides, passed with 4 Passed with 4 or less slides Number of Slides Extracted

30 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA FFPE Tissue FFPE-CB Duplicates Failed 23 Lung FFPE Samples Alk fusion pos Ret fusion pos 1 Fusion neg

31 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA Alk fusion pos Ret fusion pos Fusion neg UWHC 9 ALK fusion pos 13 Fusion neg 1 RET fusion pos 8 ALK fusions 10 fusion neg 1 RET fusion Reference lab 10 ALK fusion pos 12 Fusion neg 1 RET fusion pos 4 discordant results 2 false positive ALK fusions (FISH); NGS- was confirmed by NGS 2 false positives our NGS+ ALK, BRAF fusions (ALK- (FISH, NGS); BRAF-(NGS)) our BRAF fusion+ fails GSP2 unique start sites <3 (7 fusion spanning reads) our ALK fusion+ GSP2 unique start sites = 3 (15 fusion spanning reads)

32 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit FFPE Tissue TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA A Novel Fusion Discovery CTTNBP2-MET DQ FNA Fusion spanning reads (%): 8 (100%) 41 (41%) Unique fusion start sites: 4 16

33 Maxwell CSC FFPE RNA Purification Protocol Promega Maxwell CSC Maxwell CSC RNA FFPE Kit TNA Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA A Novel Fusion Discovery CTTNBP2-MET Unique reads supporting fusion

34 Maxwell CSC FFPE RNA Purification Protocol TNA Promega Maxwell CSC 1,3 Maxwell CSC RNA FFPE Kit 1,2,3 1 For In vitro Diagnostic Use 2 Intended for use with FFPE tissues collected from human breast, lung or colon 3 The studies shown in this presentation are research studies and are not intended for diagnostic procedures Practical: small footprint; tablet controlled; handheld barcode scanner Documentation: reagent lots, protocols, samples, runs and users Kit(s): all inclusive reagents: minimal preparation, room temperature stable Procedure: no organics, compatible w/ macrodissection, single tube process Nucleic acids: yield, 50uL eluate; FFPE: 2 slides DNA; 4 slides TNA NA performance: free of inhibitors, functional in a variety of assays (NGS) Alternate Sample Types 3 : FFPE tissue, FNA, cell blocks, bone marrow DNA: macrodissection; 4.5hrs preprocessing (4hrs 80 C); 40 min. Maxwell TNA: macrodissection; 1.5hrs preprocessing (1hr 80 C); 40 min. Maxwell

35 Archer FusionPlex System Comprehensive Thyroid and Lung FusionPlex RNA Reagents/Process: All reagents included; barcodes are separate (Illumina/Ion Torrent) PreSeq, First/Second Strand Synthesis (random priming) extra (2x) Parallel, lyophilized workflow Packaged together for 8 samples; color coded (steps) for ease of use Quality Assurance: RNA PreSeq (qpcr to evaluate RNA quality (NGS functionality) Kapa (NGS specific/separate kit) qpcr normalize library/template GSP2 (gene specific primers 2; avg. 4 controls) Unique Start Sites (>10) Fusion Unique Starts (>3); Spanning Reads (Breakpoint Coverage) Detection: Archer AMP technology: permits novel fusion partner detection Compatible with Illumina and Ion Torrent NGS chemistries Time: Post TNA (RNA) extraction, PreSeq, and then ~2 days to NGS

36 Acknowledgements Dr. Molly Accola, Ph.D UWHC Molecular Diagnostics Staff UWSMPH Pathology Chair Andreas Friedl MD UWSMPH Pathology Faculty (Drs. Darya Buehler and Scott Aesif) UW Health Pathology Residents (Dr. Richard Yang and others) Dr. Suzanne Selvaggi, MD (Director of UWHC Cytopathology) Dr. Chris Hartley, MD (Fellow in Cytopathology) UWHC Quality Specialists: Jill Palmer and Katie Dodge Doug Horejsh Herly Karlen Curtis Knox Josh Stahl Jon Bon Mike Cerney