Multiplex profiling of cancer driver mutations: Detection of ... - NanoString [PDF]

Multiplex profiling of cancer driver mutations: Detection of single nucleotide variants and small InDels from small amounts of FFPE samples on the nCounter® analysis system NanoString Technologies 530 Fairview Avenue North, Seattle, WA 98109

Cancer Genetics # 2895F The nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel Enables Routine Detection From FFPE samples

Tools and methods to perform rapid and robust multiplexed molecular profiling of formalin-fixed, paraffin-embedded (FFPE) tissue samples are critical for basic and translational cancer research. NanoString’s optical barcoding technology has been adapted to enable detection of small genomic sequence changes such as SNVs, multiple base substitution variants, and insertions and/or deletions of less than 20 bases through development of a novel class of variant-specific hybridization probes1. A test (alpha) panel of variant-specific probes that targets 26 important cancer-associated variants was developed that permits multiplex detection of SNVs from gDNA. The number of SNVs assayed was expanded to 78 then 112 during the course of this study. This research assay can be combined with other nCounter Vantage™ panels* for 3D Biology™ to enable, for example, simultaneous detection of fusion gene expression from RNA. The workflow begins with a pre-amplification of gDNA (fresh or FFPE-extracted) via multiplex PCR to enrich targets of interest. Resulting amplicons are then hybridized with the SNV panel of variant-specific nCounter probes. After hybridization, pooling with any other 3D Biology panel can be done for simultaneous quantification within a single cartridge lane on an nCounter system. SNV panel specific controls have been implemented for both thorough QC measures and proper statistical analysis. All variant-specific hybridization probes in the panel were verified to have accurate detection down to 5% variant allele fraction, with many able to go as low as 1%. As little as 5 ng of FFPE-extracted gDNA is sufficient for the assay, supporting minimal use of precious samples, such as FFPE tissues and obviating the need for microdissection. Using 5 ng, simultaneous detection of 16 variants, each present at either 2.4% or 4.8% allele fraction, was demonstrated using a blend of genomic DNA from engineered cell lines. Most importantly, SNV mutations were detectable from a range of FFPE samples that were known to harbor mutations (as low as 5% tumor) as verified by deep targeted sequencing or qPCR. As compared to the NGS data, sensitivity and specificity of variant detection are each >98% with a FDR of < 2% for the nCounter SNV assay. Significantly, we also show that the SNV detection chemistry can be combined with fusion-gene expression analysis by using an nCounter Vantage RNA Panel in a 3D Biology workflow to provide simultaneous assay of these two important classes of analytes, DNA and RNA, from limited amounts of nucleic acid.

Results shown from 5 ng of genomic DNA extracted from FFPE sections purchased from Horizon Discovery plc. and blended to contain 10 different variants ranging in frequency from 1% to 10%. All assayed variants are detected with > 95% confidence (p < 0.05 confidence threshold indicated by the dashed pink line).

0.00001

p-value 0.001 0.01

0.0001

0.1

1

Variant allele frequency 5.5%

10%

1.5%

4.5%

Probe Design for Specific SNV Detection

Capture probe

Probe T

Probe S

0.00001 KRAS_varCOSM516 KRAS_varCOSM520 KRAS_varCOSM521 KRAS_varCOSM522 KRAS_varCOSM527 KRAS_varCOSM532 KRAS_varCOSM517 KRAS_varCOSM512 KRAS_varCOSM518 KRAS_varCOSM533 KRAS_varCOSM529 KRAS_varCOSM528 EGFR_varCOSM6224 EGFR_varCOSM6213 TP53_varCOSM11081 TP53_varCOSM6549 TP53_varCOSM43871 TP53_varCOSM10817 TP53_varCOSM10662 TP53_varCOSM10656 TP53_varCOSM6530 TP53_varCOSM10705 TP53_varCOSM10733 TP53_varCOSM11066 TP53_varCOSM11089 TP53_varCOSM44571 TP53_varCOSM10742 PIK3CA_varCOSM763 PIK3CA_varCOSM760 PIK3CA_varCOSM27133 PIK3CA_varCOSM764 PIK3CA_varCOSM766 PIK3CA_varCOSM6147 PIK3CA_varCOSM12459 EGFR_varCOSM6223 EGFR_varCOSM6225 EGFR_varCOSM12416 EGFR_varCOSM12384 EGFR_varCOSM6254 EGFR_varCOSM12382 EGFR_varCOSM6255 EGFR_varNOCOSM7 EGFR_varCOSM12369 EGFR_varCOSM12370 BRAF_varCOSM476 BRAF_varCOSM478 BRAF_varCOSM475 BRAF_varCOSM6137 BRAF_varCOSM473 BRAF_varCOSM467 EGFR_varCOSM6239 EGFR_varCOSM51525 EGFR_varCOSM6253 EGFR_varNOCOSM8 EGFR_varNOCOSM9 CTNNB1_varCOSM5662 CTNNB1_varCOSM5664 CTNNB1_varCOSM5667 CTNNB1_varCOSM5730

1%

Reporter tag

60%

Variants at 1-10% allele frequencies detected from 5 ng of FFPE DNA

Introduction In a continuing effort to meet the needs of cancer researchers, NanoString Technologies has developed novel hybridization probe chemistry (‘SNV probes’) that can specifically detect single- and multi-base substitutions (SNVs and MNVs) as well as single- or multi-base insertions, deletions, or complex combinations of such genomic changes (InDels). The novel SNV assay probe design features a ‘Probe S’ that is composed to two modified oligonucleotides that are linked through a 25-30 base stem sequence but that do not otherwise complement each other; the non-complementary extensions of both oligos can be thought of as “arms”, each of which is designed to contiguously probe the DNA molecule of interest. The binding of these two arms is not independent and appears to function in a cooperative manner since the effective melting temperature of such DNA-probe complexes is demonstrably higher than the predicted melting temperature of either “arm” sequence. Notably, single nucleotide mis-matches against either arm can destabilize binding to targeted, otherwise complementary, single-stranded nucleic acid. This chemistry has now been scaled-up to permit the interrogation of >100 genomic sequence variants.

Accurate SNV Detection from a Wide Range of FFPE Qualities

Variant allele frequency

Abstract

5.25%

DNA target fragment of interest

p-value 0.001 0.01

0.0001

0.1

1

Variant allele frequency

NRAS_varCOSM583

50% 40% 30% 20% 10% 0%

NRAS_varCOSM586

1

NRAS_varCOSM585

2

3

SNV Test Panel

Each SNV Probe is an oligonucleotide-based construct with: • •

Gene

Probe S with two ‘short’ arms  base-pairs contiguously with DNA target strand Reporter tag  uses existing NanoString optical fluorescent barcodes • A single mis-match on one arm inhibits hybridization of SNV probe S • Detection of an SNV results from counting the SNV assigned barcode • A simple hybridization protocol precedes nCounter quantification • Each assay use probes for the reference and the variant allele

The nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel

APC BRAF EGFR ERBB2 FBXW7 KRAS NRAS PIK3CA TP53

SNVs Assayed 1 2 2 1 1 13 4 1 1

Total SNVs:

True Positive

KRAS_varCOSM554

Putative False Positive

KRAS_varCOSM555

3D Biology™: Workflow for Simultaneous Assay of DNA SNVs and RNA Expression

KRAS_varCOSM552 KRAS_varCOSM551 KRAS_varCOSM550 KRAS_varCOSM549 8.75%

PIK3CA_varCOSM775 TP53_varCOSM11513

Extract and quantify nucleic acids

STK11_varCOSM1523962 MET_varCOSM35468 PTEN_varCOSM5159

Pool hybridization reactions & load nCounter cartidge

Hybridize in parallel overnight (16 hr)

• This pool contains primer pairs to simultaneously pre-amplify 43 target regions • This Master Mix that permits selective multiplex amplification features uracil-N-deglycosylase and incorporation of dUTP for carry-over contamination control 3) DNA SNV Reference Sample (negative for all assayed variants) • Human DNA for Whole-Genome Variant Assessment (NIST RM 8398; genome NA12878)

FFPE-01 FFPE-02 FFPE-03 FFPE-04

4) SNV Panel TagSet • This pool contains the optically detectable molecular barcodes

FFPE-05

5) Solid Tumor SNV Panel Probe S Pool

FFPE-06 FFPE-07

• Pool of all mutant and reference-specific “two-arm” probes

6) Solid Tumor SNV Panel Probe T Pool

FFPE-08

• Pool of target-specific probes that permit automated purification for optical detection

7) Solid Tumor SNV Panel Probe M Pool

FFPE-09

• Reference-allele specific signal attenuators

FFPE-10 FFPE-11 FFPE-12

The nCounter Solid Tumor SNV Detection Workflow

FFPE-13

Day 2 (multiple cartridges per system)

Overnight

FFPE-14 FFPE-15

Load nCounter cartidge

Hybridize DNA with SNV probes (16hr)

Count on nCounter system

Obtain detection calls

FFPE-16 FFPE-17

Multiplex PCR products

FFPE-18 FFPE-19 FFPE-20 FFPE-21 FFPE-22 FFPE-23

Input: 5 ng purified genomic DNA (fresh or FFPE) Hands-on-time is approximately 30 minutes per 12 samples; 12 samples per cartridge Sample-to-answer time depends on purifying & quantifying the DNA; can be A

Fusion transcript detection results

NGS Read Depth

DNA Quality (DIN)

Variant(s) detected by nCounter

Cancer Type

Primary or Metastatic

33.6% 10.8%

4.2 3.7

Y Y

Anal Cancer Pancreatic

Distant met Distant met

3.8

Y/Y

Melanoma

Distant met

10.0%

N/A N/A 1094 / 616 N/A

2.9

Y

Colorectal

Primary

15.4% / ?

N/A

2.4

Y / Y†

Colorectal

Primary

32.6% 23.7%

N/A N/A

2.8 5.5

Y Y

Lung Melanoma

Primary Primary

46.5% / 12.3%

N/A

3.3

Y

Colorectal

Primary

55.3%

N/A

4.7

Y

Melanoma

Distant met

33.6% / 34.0%

N/A

4.4

Y

Melanoma

Distant met

14.8% 12.4%

1273 N/A

3.2 6.1

Y Y

Brain Melanoma

Primary Primary

COSM26697

17.7%

541

4.2

Y

Colorectal

Primary

COSM18848 / COSM532

8.3% / 20.0%

84 / 60

3.6

Y/Y

Colorectal

Distant met

COSM18852 / COSM521

5.4% / 10.5%

895 / 936

3.4

Y/Y

Colorectal

Distant met

APC_Q1367* / BRAF_V600E

COSM13121 / COSM476

13.4% / 14.6%

469 / 1256

3.5

Y/Y

Colorectal

Primary

KRAS_G12S / APC_Q1378* BRAF_V600E BRAF_K601E BRAF_V600K CTNNB1_S45F/ NRAS_Q61L

COSM517 / COSM18862 COSM476 COSM478 COSM473 COSM5667 / COSM583

22.6% / 11.9%

765 / 354

3.3

Y/Y

Sarcoma

Primary

5.9% 18.9% 31.1%

1151 37 952

4.7 1.7 6.9

Y Y Y

Melanoma Melanoma Melanoma

5.9% / 16.5%

781 / 924

3.2

Y/Y

CTNNB1_T41A / KRAS_G12V NRAS_Q61H NRAS_Q61R / PIK3CA_H1047R NRAS_G12D NRAS_G12C EGFR_T790M EGFR_G719A PTEN_R233* PTEN_Q245* KRAS_Q61H KRAS_Q61L KRAS_G12A KRAS_G12C KRAS_G12D KRAS_G12R KRAS_G12S KRAS_G12V KRAS_G13D PIK3CA_H1047R PIK3CA_E545Q PIK3CA_E542K PIK3CA_Q546R / KRAS_G12D PIK3CA_E545K NRAS_G12D / BRAF_D594G

COSM5664 / COSM520 COSM586 COSM584 / COSM775 COSM564 COSM562 COSM6240 COSM6239 COSM5154 COSM5159 COSM554 COSM553 COSM522 COSM516 COSM521 COSM518 COSM517 COSM520 COSM532 COSM775 COSM27133 COSM760 COSM12459 / COSM521 COSM763 COSM564 / COSM467

5.9

APC_R876* KRAS_G12V KRAS_G12D / APC_I1307K KRAS_G13D KRAS_G12C / CTNNB1_T41A KRAS_G12A NRAS_Q61K NRAS_Q61L / PIK3CA_E542K NRAS_Q61R NRAS_Q61H / NRAS_Q61R PIK3CA_H1047R BRAF_V600E APC_I1307K APC_R564* / KRAS_G13D APC_R876* / KRAS_G12D

COSMIC ID COSM18852 COSM520 COSM521 / COSM26697 COSM532 COSM516 / COSM5664 COSM522 COSM580 COSM583 / COSM760 COSM584 COSM586 / COSM584 COSM775 COSM476

FBXW7_R465C / COSM22932 / FFPE-46 EGFR_L747_S752delL COSM12382§ REATS

15.5% / 42.7%

Primary Distant met Distant met

•

The nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel assay is optimized for very small amounts (5 ng) of FFPE gDNA; thus, microdissection can be avoided in many cases and precious samples conserved.

Melanoma

Distant met

•

The nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel assay workflow is simple and requires minimal handson time.

Y/Y

Colorectal

Distant met

2.2

Y

Melanoma

Distant met

•

3D Biology Enabled: Simultaneous detection of DNA and RNA variants can be performed on nCounter systems; this is particularly powerful for combined assay of driver mutations and fusion-transcripts.

•

For more information, please visit 3d.nanostring.com

39.3% 4.8% / 24.7%

600 / 433

3.1

N‡ / Y

Breast

Primary

38.3% 9.7% 29.8% 10.4% 7.3% 6.7% 4.9% 50.6% 14.0% 13.3% 5.4% 7.8% 8.5% 5.0% 7.7% 6.7% 12.4% 9.7%

1226 N/A 316 7636 1234 760 722 332 271 578 710 424 1366 402 943 958 845 2254

4.4 5.8 2.2 5.9 6.3 4.5 5 2.4 4.2 3.9 5.4 4.1 3.7 5.5 4.6 6.2 4.9 5

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Melanoma Brain Breast Brain Sarcoma Brain Appendiceal Colorectal Breast Colorectal Appendiceal Lung Colorectal Pancreatic Colorectal Head and Neck Colorectal Sarcoma

Primary Primary Distant met Primary Primary Primary Distant met Primary Distant met Primary Primary Primary Primary Primary Distant met Primary Primary Primary

16.7% / 32.6%

245 / 463

2.6

Y/Y

Colorectal

Primary

4.8%

1157 1226 / 1534

6.2

Y

Brain

Primary

4.4

Y/Y

Melanoma

Primary

N/A

2.7

38.3% / 10.2% 8% / 2.4%

Using commercially available human genomic DNA (Horizon Discovery plc), a blend was created that carried 16 solid-tumor associated SNVs at somatic frequencies (2.4% and 4.8% in blue). Upon assay with an ‘alpha’ version of the nCounter SNV panel, all 16 variants are detectable with counts levels that are 3-fold greater than counts obtained from the negative control reference sample and with an estimated confidence of > 99%. Count-level fold-change over reference (in log2 units) is indicated by the number in each red box. The estimated count variance in each assay is reflected by the size of the boxes. The 11 reference alleles’ probe counts are grouped at right.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

@nanostringtech

© 2016 NanoString Technologies, Inc. All rights reserved. Patents pending. NanoString, NanoString Technologies, the NanoString logo, nCounter, 3D Biology, and nSolver are registered trademarks or trademarks of NanoString Technologies, Inc., in the United States and/or other countries.

Solid Tumor SNV Panel

Next-Gen Sequencing

Detected

Undetected

Detected

59

1

Undetected

1

3297

Conclusions NanoString’s new SNV probe technology enables sensitive and specific detection of somatic (5% allele frequency) mutations; these include SNVs, MNVs, and InDels.

1957 / 1259 733

33.2% / 20.5%

As proof-of-principal for a 3D Biology workflow to simultaneously detect SNVs and fusion transcripts that are associated with lung cancer, DNA was extracted from a single 10 micron section of a commercially obtained KRAS G13D-positive (COSM532) lung adenocarcinoma tissue block (Asterand Bioscience) and RNA was extracted from a single 10 micron section of a commercial ALKRET-ROS1 Fusion RNA Reference Sample (Horizon Discovery). Both types of nucleic acid were processed per the workflow above and, after parallel hybridization, pooled for simultaneous readout on an nCounter FLEX system. Analysis of the nCounter digital count data indicates the presence of the expected KRAS COSM532 SNV and the presence of the expected EML4:ALK, CCDC6:RET, and SLC34A2:ROS1 fusion transcripts. The imbalance probes show unequivocal evidence for the expression of an ALK fusion-gene, a RET fusion-gene, and a ROS1 fusion-gene.

•

References 1)

Kim, D. et al. 3D Biology™: Simultaneous single-molecule quantification of DNA (SNVs), mRNA, & proteins. Poster presented at AGBT; February 2016; Orlando, FL.

2)

Meredith, G. et al. Multiplex detection of driver mutations in lung cancer: Simultaneous assay of single nucleotide variants (SNV) and fusion-transcripts from small amounts of FFPE samples on the nCounter® analysis system; (Cancer Genetics #2894T). Presented at the 66th Annual Meeting of The American Society of Human Genetics, October 20, 2016, Vancouver, Canada).

3)

Lira, M.E., Choi, Y., Lim, S.M. et al. A single–tube multiplexed assay for detecting ALK, ROS1, and RET fusions in lung cancer. J Mol Diagn. 2014 Mar; 16(2):229-43

4)

Lovly, C., Horn, L., and Pao, W. Molecular Profiling of Lung Cancer. My Cancer Genome https://www.mycancergenome.org/content/disease/lung-cancer/

Acknowledgements Y/Y

Bladder

Met

Genomic variant positions addressed by prior targeted next generation sequencing (average read depth of >200X) and the nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel shown above. Variant detection concordance and summary statistics are shown below.

www.nanostring.com | [email protected] |

Obtain detection calls

PTEN_varCOSM5154

Variant Allele Frequency by NGS

Mutation(s) Sample ID identified by NGS

2) SNV Amplification MasterMix (5X)

Fresh or FFPE sample

Count on nCounter system

59/60 variants detected by NGS also detected by SNV assay from 46 FFPE samples

1) Solid Tumor SNV Panel PCR Primer Pool

Amplify target regions

ALK APC BRAF BRCA1 BRCA2 CTNNB1 EGFR ERBB2 FBXW7 GNA11 GNAQ JAK2

False Negative

KRAS_varCOSM553

Solid Tumor SNV Panel Gene

7

Reliable SNV Detection from FFPE Samples with the Solid Tumor SNV Subset Panel

SNV Panel reagents are provided in 7 tubes:

Extract and quantify DNA

ALK APC BRAF BRCA1 BRCA2 CTNNB1 EGFR KRAS MET NRAS PIK3CA PTEN Total SNVs:

NanoString’s commercial kit and workflow designed to enable researchers to simultaneously screen for the presence of > 100 somatic sequence variants from just 5 ng of FFPE-extracted gDNA.

Day 1 (~3 hrs)

26

SNVs Assayed 5 7 5 2 2 4 15 19 1 9 7 2

Gene

6

NRAS_varCOSM580

Extensive Content Addresses Many Key Cancer Genes Solid Tumor SNV Subset Panel

5

DNA Integrity Number (DIN)

NRAS_varCOSM584 8.75% 2.5%

4

Sensitivity Specificity Precision Accuracy FDR

98.3% 100.0% 98.3% 99.9% 1.7%

As compared to the NGS dataset: Y† = a Putative False Positive call N‡ = a False Negative call COSM12382§ = InDel detected

Data and results cited in this study were funded, in part, as follows: The Sheikh Bin Zayed Al Nahyan Foundation (1U01 CA180964), NCATS grant UL1 TR000371 (Center for Clinical and Translational Sciences). The Bosarge Foundation, The MD Anderson Cancer Center Support grant (NIH/NCI P30 CA016672), the MD Anderson Moon Shot Program; K. Chen, NCI R01 CA172652; K.M. Shaw, institutional funding from the Khalifa Foundation; M.A. Davies, NIH 1RO1 CA172670. NanoString Technologies also supports ongoing work supervised by G.B. Mills at The University of Texas MD Anderson Cancer Center. * For Research Use Only, not for use in diagnostic procedures.

P. Martin Ross1, Jinho Lee2, Jill McKay-Fleisch1, Christopher P. Vellano2, Jessica Garber1, Agda K. Eterovic2, Elizabeth Manrao1, Mike Krouse1, Joseph Phan1, Mekala Pansalawatta1, Erin Piazza1, Afshin Mashadi-Hossein1, Jason Loo1, Karen Nguyen1, Lucas Dennis1, Anisha Kharkia1, Dae Kim1, Gordon B. Mills2, Gavin Meredith1, Joseph Beechem1 1) R&D, NanoString Technologies, Seattle, WA 2) Dept. of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX

October 2016