Lab Approach for Basket Trials in Advanced Tumors Mohamed Salama - - PowerPoint PPT Presentation

Lab Approach for Basket Trials in Advanced Tumors

Mohamed Salama M.D. Professor of Pathology, University of Utah

Objectives

Discuss how to support basket trials as a pathology department Basket trials require an integrated approach that brings together microscopic work, screening and confirmation lab testing as well coordination with the clinical team

Outline

• Pathology and clinical trials design
• Basket trials can offer opportunities or challenge
• STARTRK-2 Trial as a case study

– Patients’ response – Laboratory approach

– IHC → NGS – Digital Expression Profiling (Nano string)

• Future directions and academic opportunities

Clinical Trial Paradigm

Umbrella Trials Basket Trials

VS

Umbrella Trials

Histology-specific genotyping study

Basket Trials

Histology-independent, aberration specific

• Targeted drug, develop simultaneously across organ- specific tumors, restricted

to those expressing target

• Sample sizes tiny, borrow but do not “pool” (formalizes ‘Gleevec phenomenon”)

The Logistics of Implementing Basket Trials

• To be successful, basket trials require coordination

across tumor areas – lung, GI, head and neck, hematology, sarcoma/melanoma, gyn, etc.

• Oncopolitics sets up communication barriers between

these groups

• These trials often require new testing methodologies –

for example testing heterogeneous fusions across tumor types – this takes time and money to implement

• Pathology is a central player in basket trials

STARTRK-2 Trial as an Example Case Study

• Huntsman Cancer Institute was selected as a site for

STARTRK-2 trial

• ARUP is a pathology service provider for Huntsman,

– identify fusions of NTRK, ROS1, and ALK across many tumor types

STARTRK-2 Trial

An Open-Label, Multicenter, Global Phase 2 Basket Study

• f Entrectinib for the Treatment of Patients with Locally

Advanced or Metastatic Tumors that Harbor NTRK1/2/3, ROS1, or ALK Gene Rearrangements

Entrectinib (RXDX-101): A First-in-Class Trk Inhibitor and Potential Best-in-Class ROS1 Inhibitor

♦

Most potent pan-Trk-inhibitor in clinical development with activity against most

• f the known Trk-resistant mutants

Target TrkA TrkB TrkC ROS1 ALK IC50* (nM) 1.7 0.1 0.1 0.2 1.6

Most potent, orally available pan-Trk inhibitor in clinical development

♦

30x more potent against ROS1 than crizotinib; high potency against ALK

♦

Designed to cross blood brain barrier (BBB) and to address CNS metastases, a common complication of advanced solid tumors

♦

Demonstrates inhibition of its RTK targets and down- stream effectors in the PLCγ, MAPK and PI3K/AKT pathways

♦

Entrectinib-mediated inhibition of oncogenic fusion proteins results in rapid tumor response in preclinical models and in selected patient populations

* Biochemical kinase assay

Antitumor Activity in ALK and ROS1 Inhibitor-Naïve Patients with NTRK1/2/3, ROS1, or ALK Gene Rearrangements



25 patients treated



24 patients with extracranial solid tumors, locally assessed Overall Response Rate: 19/24 (79%) NTRK patients: 3/3 (100%) ROS1 patients: 12/14 (86%) ALK patients: 4/7 (57%)

* RECIST criteria not validated in primary brain tumors (FDA-AACR Brain Tumor Endpoints Workshop 2006)

• 100
• 90
• 80
• 70
• 60
• 50
• 40
• 30
• 20
• 10

10 20 30

Sum of longest diameter, maximum decrease from baseline (%)

PD



PR CR

Data cutoff 07 March 2016

Non-Enhancing Volume (cm3) Enhancing Volume (cm3) 2

4 6 8 10 12 14 11.66 cm3 6.45 cm3 Jul 2015 Feb 2016

entrectinib initiated

1 patient with NTRK+ astrocytoma SD by RECIST* 45% by exploratory 3-D volumetric assessment

Gene Rearrangements Targeted by Entrectinib Are Present in a Large Number of Tumors

NTRK1 NTRK2 NTRK3 ROS1 ALK

NSCLC (adeno, large cell NE)

1-3% <1% <1% 1-2% 3-7%

CRC

1-2% 1% 1-2% 1-2%

Salivary gland – mammary analog secretory carcinoma [MASC]

90-100%

Salivary gland – NOS

3%

Sarcomas (including GIST)

1-9% 2-11% 2-3% 1-5%

Astrocytoma

3%

Glioblastoma

1-3% 1%

Melanoma (Spitz)

16% 17% 10%

Cholangiocarcinoma

4% 9% 2%

Papillary thyroid carcinoma

4-13% 2-14% 7%

Breast – secretory carcinoma

92%

Breast – NOS

2%

Clinical response to entrectinib in a 46 year-old male patient with NTRK1-rearranged NSCLC

Response of Brain Metastases

NTRK1 (TrkA) Rearrangements Across Tumors

TM Kinase Domain NTRK (wild-type) Signal Peptide/Extracellular Domain MPRIP-NTRK1 NSCLC IRF2BP2-NTRK1 NSCLC, PTC RFWD2-NTRK1 NSCLC TPM3-NTRK1

CRC, PTC, NSCLC, sarcoma, pediatric glioma, breast, gallbladder, cholangiocarcinoma

LMNA-NTRK1 CRC, Spitzoid melanoma, sarcoma TP53-NTRK1 Spitzoid melanoma TFG-NTRK1 PTC TPR-NTRK1 CRC, PTC NFASC-NTRK1 GBM PEAR1-NTRK1 Sarcoma, breast cancer SQSTM1-NTRK1 NSCLC, PTC, sacrcoma CD74-NTRK1 NSCLC, GBM BCAN-NTRK1 MDM4-NTRK1 RABGAP1L-NTRK1 PPL-NTRK1 CHTOP-NTRK1 ARHGEF2-NTRK1 TAF-NTRK1 CEL-NTRK1 SSBP2-NTRK1 GRIPAP1-NTRK1 LRRC71-NTRK1 MRPL24-NTRK1 Astrocytoma/GBM Breast cancer Cholangiocarcinoma PTC GBM GBM PTC Pancreatic cancer PTC NSCLC NSCLC Uterus carcinoma

STARTRK-2 Clinical Trial Testing Strategy:

IHC enrichment followed by NGS

1. IHC cocktail to detect expression in NTRK1/2/3, ROS1, ALK – a hallmark of gene rearrangement 2. Removes 50-70% of cases from further Dx consideration 1. RNA based Anchored multiplex PCR library preparation 2. Less bias for gene rearrangements

Step 1: IHC screening Step 2: Sequencing

Finding the Responders

IHC Measures Active Protein

• Oncogenic gene rearrangements need to produce active chimeric protein

(e.g., in frame). This will not be observed with typical methods, such as FISH.

• Example: Ovarian carcinoma, clear cell

ALK FISH+ (confirmed) Pan-IHC- (ALK IHC-)

Colorectal Adenocarcinoma

ALK Pan-IHC ROS1 Trk

NGS – TPM3:NTRK1 NTRK FISH+ Lee, et al, Samsung

Colorectal Adenocarcinoma

• ALK expression from gene fusion but also background Trk expression

ALK Pan-IHC ROS1 Trk

NGS – EML4:ALK ALK FISH+ Lee, et al, Samsung

Application Across Multiple Tissue Types

Site: n Neg Pos %Pos Prostate

10 8 2 20%

Colon

266 198 68 26%

Thyroid

58 40 18 31%

Lung

204 138 66 32%

Breast

47 29 18 38%

Skin

19 10 9 47%

Ovary

15 6 9 60%

Brain

9 3 6 67%

Stomach/GI

8 2 6 75%

Totals: 636 434 202 32% NGS Step 2: Fusion Detection

Colon (5.5%)

Pos Neg IHC Pos 5 31 Neg 54

Thyroid (3.5%)

Pos Neg IHC Pos 1 6 Neg 21

Lung (3%)

Pos Neg IHC Pos 2 41 Neg 31

IHC Step 1: Enrichment

Boomer, et al, (2015) AACR

NEXT GENERATION SEQUENCING (NGS)

Multiple Methods

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• With such large intronic spaces (and difficult regions within them), fusion

detection by DNA NGS can be complicated

– Lower capacity (fewer samples to multiplex to get appropriate coverage) – More complications with difficult introns

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The Challenge – Detecting Fusions in NTRK1/2/3, ROS1 and ALK Genes

NTRK1 NTRK2 NTRK3 ROS1 ALK Intron Exon NTRK2, Intron 14 ~109kb NTRK3, Intron 13 ~93kb

Advantage of RNA for Gene Rearrangements

• Gets around the intron problem – looking for exon/exon junctions

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Beadling et al J Mol Diagn 2016, 18: 165-175

Overview - NGS Assay for Rearrangements

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Total Nucleic Acid Extraction: Agencourt Formapure Kit FFPE Slides Enzymatics Sequencing on MiSeq Custom informatics

SAMPLE EXTRACTION Library Preparation Sequence/ Analyze

Microdissection

RNA

Anchored Multiplex PCR (AMP) overview

cDNA or DNA fragments

Adapter ligation End repair, d/A-tailing

Barcode

Universal primer binding site Gene Specific Primer 1 (GSP1) Partially functional MBC adapter Sequencing-ready library Nested PCR with GSP2

GSP1 GSP2 P5 Primer

Amplicon library

✗ ✗ ✗

Indistinguishable amplicons, Lost information Unique, barcoded ends AMP library Anchored Multiplex PCR Traditional Opposing Primers

Archer AMP vs Opposing Primers (Ampliseq/Oncomine)

*Courtesy: ArcherDx

Gene Rearrangements Detected in Association with STARTRK-2

Gene rearrangements detected through Ignyta and other collaborative diagnostic partnerships

Head & Neck Sarcomas Gastrointestinal

• Salivary gland cancer (MASC): NTRK
• Sarcoma: ALK, NTRK
• CRC: NTRK, ROS1, ALK
• Squamous cell: NTRK, ALK
• Soft tissue angiosarcoma: ROS1, ALK - CRC (appendiceal): NTRK
• Papillary thyroid: NTRK, ROS1, ALK
• Infatile fibrosarcoma: NTRK
• Cholangiocarcinoma: NTRK

Thoracic

• Gliosarcoma: NTRK
• Gastroesophageal: NTRK, ROS1
• NSCLC (adeno): NTRK, ROS1, ALK
• Uterine adenosarcoma: NTRK
• Pancreas: NTRK, ALK
• NSCLC (squamous): NTRK, ROS1, ALK
• Liposarcoma: NTRK
• Bile duct: NTRK
• Small cell carcinoma: ROS1, ALK
• Ewing's sarcoma: ALK
• Stomach adenocarcinoma: NTRK

CNS

• Stromal sarcoma: NTRK

Genitourinary

• Gliosarcoma: NTRK

Breast

• Renal cell carcinoma: ALK
• Astrocytoma: NTRK
• Breast (secretory): NTRK

Other

• Glioblastoma multiforme: ROS1
• Breast: ROS1, ALK
• Melanoma: ROS1

Gynecological Hematological Malignancies

• Neuroendocrine: NTRK
• Uterine adenosarcoma: NTRK
• Anaplastic lymphoma: ALK
• Soft tissue myofibroblastic tumor: ROS1
• Ovarian/fallopian epithelial: NTRK, ALK
• Pediatric ALL: NTRK
• Peripheral nerve sheath tumor: NTRK
• Uterine Leiomyosarcoma: ALK
• Mutliple myeloma: NTRK

LASTLY - NANOSTRING

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Gene 1 Gene 2 Gene 3

Nanostring Technology

Direct measurement of RNA expression (no PCR) Can be used in degraded samples (FFPE)

Junction Probe

• Int. Jnl. Lab. Hem. 2015, 37, 690–698

5’/3’ Positional Imbalanced Probe Gene Expression Design:

♦

No partner information

EML4-ALK (exon 20)

5’/3’ Positional Imbalanced Probe Gene Expression Design:

♦

Wild-type expression can complicate analysis

• Difficult to identify the fusion threshold with a background
• Use of fusion specific probes can assist for known partners

EZR:ROS1 (exon 34)

Exon 1 Exon 43

Lira ME etal. J Mol Diagn. 2014; 16(2):229-43.

Summary

• Basket trials provide opportunity for integration of

platforms; (IHC, microscopic identification of secretory tumor) and (Nanostring, Illlumina NGS) to work together

• Pathologists role is essential in basket trials – helping
• ncologists identify patients.
• Trials like STARTRK-2 present opportunities to advance

technology and innovation

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Questions?