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 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 Data cutoff 07 March 2016 25 patients treated 30 Sum of longest diameter, maximum decrease from baseline (%) 20 PD 24 patients with extracranial solid tumors, locally assessed 10 0 Overall Response Rate: 19/24 (79%) -10 NTRK patients: 3/3 (100%) -20 ROS1 patients: 12/14 (86%) PR -30 -40 ALK patients: 4/7 (57%) -50 1 patient with NTRK + astrocytoma -60 -70 SD by RECIST* -80 45% by exploratory 3-D volumetric -90 assessment -100 CR entrectinib initiated 14 11.66 cm 3 12 Enhancing Volume (cm 3 ) 10 Non-Enhancing Volume (cm 3 ) 8 6.45 cm 3 6 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 90-100% secretory carcinoma [MASC] 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 NTRK (wild-type) Signal Peptide/Extracellular Domain Kinase Domain TM CRC, PTC, NSCLC, sarcoma, pediatric glioma, TPM3-NTRK1 breast, gallbladder, cholangiocarcinoma LMNA-NTRK1 CRC, Spitzoid melanoma, sarcoma NSCLC, PTC, sacrcoma SQSTM1-NTRK1 CRC, PTC TPR-NTRK1 Sarcoma, breast cancer PEAR1-NTRK1 CD74-NTRK1 NSCLC, GBM IRF2BP2-NTRK1 NSCLC, PTC MPRIP-NTRK1 NSCLC RFWD2-NTRK1 NSCLC Spitzoid melanoma TP53-NTRK1 PTC TFG-NTRK1 NFASC-NTRK1 GBM BCAN-NTRK1 Astrocytoma/GBM Breast cancer MDM4-NTRK1 Cholangiocarcinoma RABGAP1L-NTRK1 PPL-NTRK1 PTC GBM CHTOP-NTRK1 GBM ARHGEF2-NTRK1 PTC TAF-NTRK1 Pancreatic cancer CEL-NTRK1 PTC SSBP2-NTRK1 GRIPAP1-NTRK1 NSCLC LRRC71-NTRK1 Uterus carcinoma MRPL24-NTRK1 NSCLC
STARTRK-2 Clinical Trial Testing Strategy: IHC enrichment followed by NGS Step 2: Sequencing Step 1: IHC screening 1. IHC cocktail to detect expression 1. RNA based Anchored in NTRK1/2/3, ROS1, ALK – a multiplex PCR library hallmark of gene rearrangement preparation 2. Removes 50-70% of cases from 2. Less bias for gene further Dx consideration 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 NGS Step 1: Enrichment Step 2: Fusion Detection Site: n Neg Pos %Pos Colon Pos Neg Prostate (5.5%) 10 8 2 20% Pos 5 31 Colon 266 198 68 26% IHC Neg 0 54 Thyroid 58 40 18 31% Lung 204 138 66 32% Thyroid Pos Neg (3.5%) Breast 47 29 18 38% Pos 1 6 Skin 19 10 9 47% IHC Neg 0 21 Ovary 15 6 9 60% Brain 9 3 6 67% Lung Pos Neg (3%) Stomach/GI 8 2 6 75% Pos 2 41 Totals: 636 434 202 32% IHC Neg 0 31 Boomer, et al, (2015) AACR
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 Intron NTRK1 Exon NTRK2, Intron 14 ~109kb NTRK2 NTRK3, Intron 13 ~93kb NTRK3 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 EXTRACTION FFPE Slides Total Nucleic Acid Extraction: Agencourt Formapure Kit Microdissection Library RNA Preparation Sequence/ Sequencing on MiSeq Enzymatics Analyze Custom informatics 27
Anchored Multiplex PCR (AMP) overview cDNA or DNA fragments End repair, d/A-tailing Partially functional MBC adapter Adapter ligation Barcode P5 Primer Universal primer binding site Gene Specific Primer 1 (GSP1) GSP1 GSP2 Nested PCR with GSP2 Sequencing-ready library
Archer AMP vs Opposing Primers (Ampliseq/Oncomine) Traditional Anchored Opposing Primers Multiplex PCR Amplicon library AMP library ✗ ✗ ✗ Indistinguishable amplicons, Unique , barcoded ends 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
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