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

GEP

ABC / GCB..

2000 Epigenomics, miRNA NGS mutations 2009 2019 Sanger 1977

Issues in 2019?

• 1. Implementation of high throughput technologies in

the routine worflow of diagnostic laboratories

• 2. Transfer genomic information into the medical care

Morphology Cytogenetics Immunophenotype

Material for lymphoma diagnosis

Flow Cytometry

Molecular

PCR, RT-PCR, PCR-Q, NGS In situ hybridization 2019 2000 CCND1

Chromosomal structural variations (SVs) evaluation

1. Diagnosis confirmation 2. Identification of chromosomal alterations with prognostic implication 3. Determine treatment

• ptions

Burkitt lymphoma Del 1p36, t(14;18) negative FL

Katzenberger et al, Blood 2009

WHO 2016 - Mature B-cell neoplasms

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

WHO 2016

DLBCL Blastoid Intermediate DLBCL/BL

Cytogenetic evaluation of hematologic disease

1. Diagnosis confirmation 2. Identification of chromosomal alterations with prognostic implication 3. Determine treatment

• ptions: selection of

targeted therapies

Copie-Bergman et al, Blood 2015

1. Diagnosis confirmation 2. Identification of chromosomal alterations with prognostic implication 3. Determine treatment

• ptions

Prognostic Value of Translocation t(11;18) in Tumoral Response of Gastric MALT lymphoma to Oral Chemotherapy

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

Cytogenetic evaluation of hematologic disease

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

• RT-PCR (fusion transcripts of API2-MLT, cycline D1,…. )
• immunohistochemistry (ex: cycline D1, ALK, …)

➢ Next generation sequencing ?

CCND1

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

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

Tumour heterogeneity

FISH Protocol

Day 1

• Preparation of sections (FFPE, 3µm)

deparaffinization, pre-treatment..

• 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)

J Hematopathol 2008, 1:119-126.

FISH probes

Break-apart probes Dual-Fusion probes

breakpoint region

Gene A

breakpoint region

Gene B

breakpoint region

Gene A

Ventura et al, Journal of Molecular Diagnostics 2006

Enumeration probes

1p36/1q25 1p36 1q25

FISH limitations and pitfalls

1- FISH is targeted to the detection of specific abnormalities 2- FISH probes selection:

ex=MYC breakapart

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 Munoz Marmol et al, Histopathology 2013

First MYC ba probe: negative Second MYC ba probe positive

Next Generation Sequencing (NGS) and chromosomal SVs evaluation

Meyerson et al, vol 11, 2010

NGS for SVs detection

• 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)

Wet bench steps for capture-based sequencing Yohe et al, Arch Pathol Lab Med 2017

Targeted hybrid capture-based assay

+ 3- Capture Hybrids on magnetic beads (target enrichment) + 4- Clonal amplification of captured DNA sequences 1- Shear DNA 2- Hybridization with custom made biotinylated probes

DNA fragments of 150-300 pb End repair Ligation to paired-end adapters

5- Sequence: need for high capacity sequencing system and dedicated SVs detection tools

DNA extraction from FFPE tissue samples

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

• Targeted hybrid capture assay
• f MYC, BCL2, BCL6, IG loci

SVs detection tools: deStruct and DELLY

Identify MYC breaks at bp resolution and MYC partner gene in 88 cases

In 24 cases (21%), no MYC-R was detected by NGS: technical issues, biological variability (eg tumor heterogeneity), design of the probes which does not encompass all breakpoints at the loci of interest.

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

Interphase FISH assays versus NGS for Chromosomal SVs evaluation