Mark&R.&Miglarese,&Ph.D.& VP&R&D& · PDF file©2017&Caris&Life&Sciences&...

©2017 Caris Life Sciences 1

Valida5on of a Microsatellite Instability Assay by NGS

Mark R. Miglarese, Ph.D. VP R&D

©2017 Caris Life Sciences

Caris Life Sciences®

•  Headquarters: Irving, Texas •  Laboratory: Phoenix, Arizona

–  66,000 sq. O. oncology-‐focused –  ISO 15189, CLIA, CAP, NY State and CE Mark approved/cer5fied

•  Profiled 120,000+ pa9ents –  7,000+ physicians –  All 50 states –  63 countries

•  Laboratory staff –  Pathologists –  Molecular Gene5cists –  Consul5ng Medical Oncologists –  MD and/or PhD literature evidence review team –  ~100 trained molecular & laboratory technicians

2

230,000+ tests performed in 2016


Comprehensive Technological Arsenal Fuels Inven5on Across a Broad Range of Molecular Biology

Comprehensive Technology Suite • Sequencing (Next-‐Gen, Whole Exome, Sanger, Pyro)

• Nanostring • PCR (ddPCR, nanoliter scale PCR, qPCR, RT-‐PCR) • Laser Capture Microdissec5on • In Situ Hybridiza5on • Histology, special stains • Immunohistochemistry • Autostaining (DAKO, Ventana) • Slide imaging • Immunoprecipita5on, immunoblohng • Cell culture • ELISA • Robo5cs

• Protein cross-‐linking • Microarray • Mass Spec (Maldi TOF, Quan5ta5ve proteomics)

• HPLC, FPLC • Polyligandhistochemistry • Luminex • Surface plasmon resonance • Dynamic Light Scajering • Fluorescent Ac5vated Cell Sor5ng • In vitro pharmacology • Oligo synthesis (na5ve or modified) • Nanosight • Exosome isola5on and characteriza5on • Liposomal formula5ons

Mul9-‐Disciplinary Exper9se • Bioinforma5cians • Mathema5cians/Sta5s5cians • Medical Oncologists • Molecular Biologists • Molecular Gene5cists • Molecular Pathologists • Research Scien5sts

3


Caris Clinical Tes5ng Menu

4

Next Gen DNA Sequencing Illumina NextSeq System

ü  592 full genes sequenced

ü  Includes all SNVs and indels on guidelines

ü  Includes nearly all markers included in clinical trials

Also reported Ø  Total Muta5onal Load

Ø  Copy Number Varia5on on 442 genes

Ø  Micro Satellite Instability

Next Gen RNA Sequencing Illumina MiSeq System

ü  53 genes sequenced

ü  Novel transloca5on detec5on independent of intronic breakpoint

ü  ALK transloca5on ü  ROS1 transloca5on ü  RET transloca5on ü  NTRK 1-‐3 transloca5ons ü  CMET exon 14 skipping ü  EFGFv3 ü  BRAF transloca5on ü  RSPO3 transloca5on

Protein Expression Ventana & Dako Immunohistochemistry

ü  25 lineage specific IHC

ü  Lung ü  PDL-‐1 (22c3) ü  ALK ü  PTEN ü  RRM1 ü  TOPO1 ü  TS ü  TUBB3

ü  CRC ü  MMR markers

ü  Breast ü  ER, PR, AR, Her2

hjp://www.carismolecularintelligence.com/tumor-‐profiling-‐menu/individual-‐tumor-‐profiling-‐menu-‐usa/


Low High

5

Iden5fying key molecular features that bring value requires understanding the complexity of the system we are working within

Complexity & Func9onal Diversity


Caris CMI Testing for Immunotherapies

6

©2017 Caris Life Sciences 7

The World just Changed – FDA approved pembro across all solid tumors that are MSI-‐High


MSI leads to an increase in the number of neoan5gens and increases the likelihood of immune recogni5on

8 Nature Biotechnology


Responses observed in a large propor5on of pa5ents

9 (Le et al. Science 2017)


Significant increase in survival was observed across MSI-‐High pa5ents



Tradi5onal approach employs PCR to compare tumor to normal across 5 loci

11


Distribu5on of 27,039 polymorphic microsatellite markers across the human genome

12 Tamiya (2005). Human molecular gene5cs. 14. 2305-‐21. 10.1093/hmg/ddi234.


MSI Valida5on Process

13

Compare NGS data to PCR (comparing cancer and normal 5ssue) on ~2200 pa5ents

Algorithm Training: Maximize Sensi5vity, Specificity, NPV, and PPV

Test on 200 Naïve pa5ents and compare to MSI by PCR and MMR by IHC

All (2196 Pa9ents) Sensi5vity Specificity PPV NPV

95.8% 99.4% 94.5% 99.2%

CRC (1198 Pa9ents) Sensi5vity Specificity PPV NPV

100.0% 99.9% 98.7% 99.6%


Distribu5on of in-‐dels across 5K pa5ents

MSI

Number of loci

Freq

uenc

y of

pat

ient

s

20 40 60 80 100 120

010

020

030

0

14



●

●● ●● ●● ●●●● ●●●● ●●● ● ●●● ●●● ●

● ●● ●

● ●●● ●●● ●●● ● ●● ●●● ● ●●●● ● ● ● ●● ● ● ● ●●● ●●● ●●● ●●● ● ●● ●● ● ●●●● ●●● ●● ●●● ●● ●● ●● ●●●● ●● ●●● ●● ●●●● ● ● ● ●●● ● ● ●●●

●●

●

●●● ●● ●● ● ●● ●● ● ●● ●● ●● ●● ● ●● ●●● ● ●● ●● ●● ●●●●● ●●●● ●●● ● ●● ●● ● ● ● ● ●●● ● ●●● ● ●●● ●●●●● ●● ●●● ●●● ●● ● ●●● ●● ● ● ●●● ●● ● ●● ●●● ●●● ● ●● ● ●●● ●● ●● ●● ● ● ●●●

●● ●● ● ● ●● ● ●●● ● ●●●● ●●

● ● ●●●●●

●● ●● ●●●● ●●●

●● ●

● ●● ●●

●

● ●

●●● ●●● ●●●● ●● ●● ●● ●●●● ●● ● ●●● ● ●●●● ●●●● ● ● ●● ●●●●● ●●

●●

●●●●●● ●

●● ● ●● ●●

●

●

● ●● ●● ● ● ●● ●● ● ● ●●● ●

● ●●● ●●●● ●● ● ●● ●●● ●● ●● ●●●● ●●● ●●● ●●● ●● ●

●● ●● ● ●●● ●● ● ● ●●●●●

●● ●● ●●

● ●

●●●● ● ● ●● ●

●● ●●● ●●●

●

●

Acute myeloid leukemia (AML) Bladder Cancer

Breast CarcinomaCholangiocarcinoma

Colorectal AdenocarcinomaEsophageal and Esophagogastric Junction Carcinoma

Extrahepatic Bile Duct AdenocarcinomaFemale Genital Tract Malignancy

Gastric AdenocarcinomaGastrointestinal Stromal Tumors (GIST)

GlioblastomaHead and neck Squamous Carcinoma

Kidney CancerLiver Hepatocellular Carcinoma

Low Grade GliomaLung Bronchioloalveolar carcinoma (BAC)Lung Non−small cell lung cancer (NSCLC)

Lung Small Cell Cancer (SCLC)Lymphoma

Male Genital Tract MalignancyMalignant Solitary Fibrous Tumor of the Pleura (MSFT)

MelanomaMultiple Myeloma

Neuroendocrine tumorsNodal Diffuse Large B−Cell Lymphoma

Non Epithelial Ovarian Cancer (non−EOC)None Of These Apply

Ovarian Surface Epithelial CarcinomasPancreatic Adenocarcinoma

Prostatic AdenocarcinomaRetroperitoneal or Peritoneal Carcinoma

Retroperitoneal or Peritoneal SarcomaSmall Intestinal Malignancies

Soft Tissue TumorsThymic CarcinomaThyroid Carcinoma

Uveal Melanoma

50 100

150

200

250

300

350

NGS MSI Count per Sample


408 194 93

46 228

1417

9

N=8952

All Lineages

TML

MSI

PDL1

Rela5onship between MSI, TML, PDL-‐1

17

3% of Pa5ents are MSI unstable

17.6% of Pa5ents are PDL-‐1 posi5ve

8.1% of Pa5ents are TML high


MSI is highly correlated to TML in Colorectal Cancer Pa5ents

18


MSI-‐NGS vs. TML – rela5onship depends on lineage

19


Rela5onship of other immunotherapy markers to MSI-‐NGS

20


Frequency of MSI across lineages


Stage 4

Stage 3


Thank you!

22

Date post:	16-Mar-2018
Category:	Documents
Upload:	lynhu
View:	218 times
Download:	3 times

Mark&R.&Miglarese,&Ph.D.& VP&R&D& · PDF file©2017&Caris&Life&Sciences&...

Documents