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 Basket Trials Umbrella 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 of 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, orally available pan-trk inhibitor in clinical development Target TrkA TrkB TrkC ROS1 ALK IC50* (nm) 1.7 0.1 0.1 0.2 1.6 Most potent pan-trk-inhibitor in clinical development with activity against most of the known Trk-resistant mutants 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 downstream 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
Sum of longest diameter, maximum decrease from baseline (%) Antitumor Activity in ALK and ROS1 Inhibitor-Naïve Patients with NTRK1/2/3, ROS1, or ALK Gene Rearrangements 25 patients treated 30 Data cutoff 07 March 2016 24 patients with extracranial solid tumors, locally assessed Overall Response Rate: 19/24 (79%) NTRK patients: 3/3 (100%) ROS1 patients: 12/14 (86%) 20 10 0-10 -20-30 PD PR ALK patients: 4/7 (57%) -40-50 1 patient with NTRK+ astrocytoma -60 SD by RECIST* 45% by exploratory 3-D volumetric assessment Enhancing Volume (cm 3 ) Non-Enhancing Volume (cm 3 ) 14 12 10 8 6 entrectinib initiated 11.66 cm 3 6.45 cm 3-70 -80-90 -100 CR 4 2 0 Jul 2015 Feb 2016 * RECIST criteria not validated in primary brain tumors (FDA-AACR Brain Tumor Endpoints Workshop 2006)
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 Signal Peptide/Extracellular Domain CRC, PTC, NSCLC, sarcoma, pediatric glioma, breast, gallbladder, cholangiocarcinoma CRC, Spitzoid melanoma, sarcoma NSCLC, PTC, sacrcoma CRC, PTC Sarcoma, breast cancer NSCLC, GBM NSCLC, PTC NSCLC NSCLC Spitzoid melanoma PTC GBM Astrocytoma/GBM Breast cancer Cholangiocarcinoma PTC GBM GBM PTC Pancreatic cancer PTC NSCLC Uterus carcinoma NSCLC TM Kinase Domain NTRK (wild-type) TPM3-NTRK1 LMNA-NTRK1 SQSTM1-NTRK1 TPR-NTRK1 PEAR1-NTRK1 CD74-NTRK1 IRF2BP2-NTRK1 MPRIP-NTRK1 RFWD2-NTRK1 TP53-NTRK1 TFG-NTRK1 NFASC-NTRK1 BCAN-NTRK1 MDM4-NTRK1 RABGAP1L-NTRK1 PPL-NTRK1 CHTOP-NTRK1 ARHGEF2-NTRK1 TAF-NTRK1 CEL-NTRK1 SSBP2-NTRK1 GRIPAP1-NTRK1 LRRC71-NTRK1 MRPL24-NTRK1
STARTRK-2 Clinical Trial Testing Strategy: IHC enrichment followed by NGS Step 1: IHC screening Step 2: Sequencing 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
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 Pan-IHC- (ALK IHC-) ALK FISH+ (confirmed)
Colorectal Adenocarcinoma Pan-IHC ALK NTRK FISH+ NGS TPM3:NTRK1 ROS1 Trk Lee, et al, Samsung
Colorectal Adenocarcinoma ALK expression from gene fusion but also background Trk expression Pan-IHC ALK ALK FISH+ NGS EML4:ALK ROS1 Trk Lee, et al, Samsung
Application Across Multiple Tissue Types IHC Step 1: Enrichment 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% Boomer, et al, (2015) AACR IHC IHC IHC NGS Step 2: Fusion Detection Colon (5.5%) Pos Neg Pos 5 31 Neg 0 54 Thyroid (3.5%) Pos Neg Pos 1 6 Neg 0 21 Lung (3%) Pos Neg Pos 2 41 Neg 0 31
Multiple Methods NEXT GENERATION SEQUENCING (NGS) 22
The Challenge Detecting Fusions in NTRK1/2/3, ROS1 and ALK Genes 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 NTRK1 NTRK2 NTRK3 NTRK2, Intron 14 ~109kb NTRK3, Intron 13 ~93kb Intron Exon ROS1 ALK 23
Advantage of RNA for Gene Rearrangements Gets around the intron problem looking for exon/exon junctions 24
Beadling et al J Mol Diagn 2016, 18: 165-175
Overview - NGS Assay for Rearrangements SAMPLE FFPE Slides EXTRACTION Total Nucleic Acid Extraction: Agencourt Formapure Kit Microdissection Sequence/ Analyze Sequencing on MiSeq Custom informatics Library Preparation Enzymatics RNA 27
Anchored Multiplex PCR (AMP) overview cdna or DNA fragments End repair, d/a-tailing Adapter ligation Partially functional MBC adapter P5 Primer Barcode Universal primer binding site GSP1 Gene Specific Primer 1 (GSP1) GSP2 Nested PCR with GSP2 Sequencing-ready library
Archer AMP vs Opposing Primers (Ampliseq/Oncomine) Anchored Multiplex PCR Traditional Opposing Primers AMP library Amplicon library Unique, barcoded ends Indistinguishable amplicons, Lost information *Courtesy: ArcherDx
Gene Rearrangements Detected in Association with STARTRK-2 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 Gene rearrangements detected through Ignyta and other collaborative diagnostic partnerships
LASTLY - NANOSTRING 31
Nanostring Technology Gene 1 Gene 2 Gene 3 Direct measurement of RNA expression (no PCR) Can be used in degraded samples (FFPE)
Int. Jnl. Lab. Hem. 2015, 37, 690 698 Junction Probe
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 Exon 1 Exon 43 EZR:ROS1 (exon 34) 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 oncologists identify patients. Trials like STARTRK-2 present opportunities to advance technology and innovation 36
Questions?