Traditional FISH or NGS approach? Pr Christiane Copie-Bergman, MD, - PowerPoint PPT Presentation

Traditional FISH or NGS approach? Pr Christiane Copie-Bergman, MD, PhD Département de Pathologie, APHP, INSERM U955 Université Paris Est Créteil (UPEC) Hôpital Henri Mondor Créteil, France Symposium Haematopathology: Technical advances in lymphoma diagnosis- Session date: Monday, 9 September 2019

• I declare no competing financial interests

WHO classification: the concept of clinicopathologic entities • Identify distinct clinicopathologic entities based on a combination of clinical features, morphology, immunophenotype , molecular and cytogenetics findings.

Genomic era of hematopathology Epigenomics, miRNA GEP NGS ABC / GCB.. mutations Sanger 1977 2000 2009 2019

Issues in 2019? 1. Implementation of high throughput technologies in the routine worflow of diagnostic laboratories 2. Transfer genomic information into the medical care

Material for lymphoma diagnosis CCND1 2000 2019 Immunophenotype Morphology Flow Cytometry Molecular Cytogenetics PCR, RT-PCR, PCR-Q, NGS In situ hybridization

Chromosomal structural variations (SVs) evaluation 1. Diagnosis confirmation Burkitt lymphoma 2. Identification of chromosomal alterations with prognostic implication 3. Determine treatment Del 1p36, options t(14;18) negative FL Katzenberger et al, Blood 2009

WHO 2016 - Mature B-cell neoplasms

WHO 2016 High grade B-cell lymphoma « double/triple hit » with MYC and BCL2 (or BCL6 ) R* (HGBL-DH/TH) DLBCL Intermediate Blastoid DLBCL/BL

Cytogenetic evaluation of hematologic disease 1. Diagnosis confirmation 2. Identification of chromosomal alterations with prognostic implication 3. Determine treatment options: selection of targeted therapies Copie-Bergman et al, Blood 2015

Cytogenetic evaluation of hematologic disease Prognostic Value of Translocation t(11;18) in Tumoral Response of Gastric MALT lymphoma to Oral 1. Diagnosis confirmation Chemotherapy 2. Identification of chromosomal alterations with prognostic implication 3. Determine treatment options Kaplan-Meier curve for event-free survival comparing t(11;18)-negative patients with t(11;18)-positive patients after 1 year of treatment with chlorambucil and after long-term follow-up Levy, M. et al. J Clin Oncol 2005; 23:5061

Chromosomal Structural variations (SVs) in cancer Fröhling et al, N Engl J Med 2008;359:722-34

Chromosomal Structural variations (SVs) in cancer Fröhling et al, N Engl J Med 2008;359:722-34

Methods for chromosomal SVs evaluation? ➢ Conventionnal cytogenetics Fresh material – labour intensive, time consuming need for dividing cells ➢ PCR (DNA) poor sensitivity in some diseases due du variability in breakpoints ➢ Interphase FISH assay Robust, high sensitivity, no need for vital, growing cells ➢ Identification of the consequence(s) of a translocation CCND1 - RT-PCR (fusion transcripts of API2- MLT, cycline D1,…. ) - immunohistochemistry (ex: cycline D1, ALK, …) ➢ Next generation sequencing ?

Fluorescent in situ hybridization ➢ Powerful and robust technique for identification of chromosomal alterations: translocations, deletion and/or numerical abnormalities ➢ Can be applied to formalin-fixed and paraffin-embedded (FFPE) tissues ➢ FISH is superior to PCR for the detection for example of BCL2 and CCND1 breaks ➢ Short turnaround time, which is usually in the order of 2 working days but which may be reduced to 3 hours by new probes ➢ Whole slide imaging is a robust alternative to traditional fluorescent microscopy for FISH (Laurent C, Human Pathol 2013) ➢ Detect the cytogenetic abnormalities in situ in the histopathological context

35 y woman, mesenteric lymph node CD5 CD20 CD10 BCL2

FISH BCL2

Tumour heterogeneity FL 3B BCL2/BCL6 HGBL-DH MYC/BCL2 , DLBCL morphology, GC DLBCL nGC BCL2/BCL6

FISH Protocol Day 1 • Preparation of sections (FFPE, 3µm) deparaffinization, pre-treatment.. J Hematopathol 2008, 1:119-126. • Pepsin digestion: 10 minutes • Hybridization with DNA FISH probes co-denaturation 5min 82°C, hybridization 14-20h 45°C Day 2 - Stringent wash, dehydratation ,… → Interpretation … Imprints Suspensions of nuclei Tissue sections: frozen, FFPE (TMA)

FISH probes Break-apart probes Dual-Fusion probes Gene A Gene A breakpoint breakpoint region region Gene B breakpoint region Ventura et al, Journal of Molecular Diagnostics 2006

1p36 1q25 Enumeration probes 1p36/1q25

FISH limitations and pitfalls 1- FISH is targeted to the detection of specific abnormalities Munoz Marmol et al, Histopathology 2013 2- FISH probes selection: ex=MYC breakapart First MYC ba probe: Second MYC ba probe negative positive King RL et al, Haematologica 2019 Chong LC. et al. Blood Adv 2018 3-Cryptic variants of MYC translocations May PC, Cancer Genetics and Cytogenetics, 2010

Next Generation Sequencing (NGS) and chromosomal SVs evaluation Meyerson et al, vol 11, 2010

• Two major approaches to detect translocations - Sequence DNA using capture method +++ - Sequence RNA (cDNA) by amplification based method (requires that a fusion protein is present) NGS for SVs detection Wet bench steps for capture-based sequencing Yohe et al, Arch Pathol Lab Med 2017

Targeted hybrid capture-based DNA extraction from FFPE tissue samples assay DNA fragments of 150-300 pb 1- Shear DNA End repair Ligation to paired-end adapters 2- Hybridization with custom made biotinylated probes + 3- Capture Hybrids on magnetic beads (target enrichment) + 4- Clonal amplification of captured DNA sequences 5- Sequence: need for high capacity sequencing system and dedicated SVs detection tools

Lauren C. Chong et al. Blood Adv 2018;2:2755-2765 • 112 tumors with DLBCL morphology and MYC-R • 2 FISH MYC breakapart assays: wide gap and narrow gap probes In 24 cases (21%), no MYC-R was detected by NGS: technical issues, • Targeted hybrid capture assay biological variability (eg tumor heterogeneity), design of the probes of MYC, BCL2, BCL6, IG loci which does not encompass all breakpoints at the loci of interest. SVs detection tools: deStruct and DELLY Identify MYC breaks at bp resolution and MYC partner gene in 88 cases

Interphase FISH assays versus NGS for Chromosomal SVs evaluation FISH NGS Advantages Robust, easily applicable technique Detection of all types of genomic alterations FFPE, no need of fresh tissue Breakpoints are characterized at single nucleotide Short turnaround /Digital slides resolution IN SITU Detection of unknown translocation partners Limitations Aimed at the most common breakpoints Occurrence of Polymerase errors during library associated with specific balanced construction translocations Preferential amplification of certain fragments in the library population May overlook additional Chr SVs Overlook breakpoints in spaces not captured by the designed probes Need for additional assays to identify High capacity sequencing systems and dedicated translocation partners bioinformatic algorithms Lack the resolution to identify the precise Dedicated bioinformatics personnel to maintain a clinical location of breakpoints NGS service Costs/ Cost ~ 95€ /probe Cost ~ 400- 1500€ + cost of validation turnaround 3h-2 days 1-2 weeks time Multiple samples can be pooled and sequenced together decreasing the sequencing cost