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Array-CGH and FISH applications for diagnosis and study of gliomas
MA. Belaud-Rotureau
Cytogenetic and Cellular Biology Laboratory
Faculty of Medicine and Rennes University Hospital
Faculté de Médecine
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• Chromosome numerical aberrations / segmental alterations
• Restricted to tumor cells selective advantage
Spécific aberrations (bcr-abl) or not (-3, +8, +7, …)
Gains: oncogenes (amplification)
Losses: Tumor Supressor Genes
Translocations gene overexpression / fusion genes
… mutations …
Clinical impact: - Diagnosis
- Prognosis
- Treatment response
Cancer
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Gliomes
• Impact pronostic des co-délétions 1p36 et 19q13
− Chimiosensibilité (réponse PCV)
− Survie globale plus longue
• Diagnostic histologique difficile
O OA A
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Asolescents and young adults
Adults > 50 years
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Asolescents and young adultsYoung adults
Improved outcome
Adults > 50 years
Paediatrics
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Gliomas
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Clinical impact of a-CGH analysis
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Resolution
Molecular biology
Molecularcytogenetics
Conventionalcytogenetics
Genome analysis
Sanlaville 2014 from http://www.calender-science.com/Cytogenetique-1.pdf
Karyotype
High resolution Karyotype
CGH
Which technique to be chosen?
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1 – Hybridation in situ en fluorescence : FISH
Hybridation d’une séquence d’ADN cible par des sondes
ADN marquées par un fluorochrome
1 - Marquage de la sonde
2 - Dénaturationde l’ADN et de la sonde
3 - Hybridation
4 - Lecture
Principe de la FISH
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FISH analysisActa Neuropathologica 2006
Del 1p36
Del 19q13Contrôle Del 1p36Del 1p36
Del 19q13
Del 19q13ContrôleContrôle
Del 1p
Del 19q
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• Routine screening
Acquisition stationSlide charger
Automatic nuclei segmentation
Image analysis
FISH analysis
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BioView’s Solution
H&E / IHC region of interest selection can be performed from any location via standard web browsers on a digital slide
H&E digitally review and marked using BioView’s SoloWeb application
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BioView’s Solution
Integrated solution to allow matching between H&E/IHC and FISH slides prepared on parallel sections
FISH analysis is performed according to pathologist guidance
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System Typical Layout
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Comparative Genomic Hybridization (CGH)
Technical steps
DNA extraction
Labelling
Scanning
analysis
Cy3 Cy5
Test Réf.
Metaphasis (Kallioniemi et al., 1992)
Hybridization(metaphasis)
Resolution: 10 – 20 Mb
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Comparative Genomic Hybridization (CGH)
High resolution
http://www.helixio.fr/contenu/support-fr/technologies-fr
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Resolution : number and distribution of clones
Metaphasis : Karyotype bands (5-10 Mb)
BACs : ~ 170 kb
Oligonucleotides
Comparative Genomic Hybridization (CGH)
180K : 30-50kb resolution200x higher than karyotype
Sanlaville 2014 from http://www.calender-science.com/Cytogenetique-1.pdf
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Data analysis
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Array-CGH for tumor classification in routine
• Genomic copy number profiles can distinguish distinct subgroups within
histologically defined disease entities
• Tumor type specific copy number patterns can be used for efficient tumor
classification and patient stratification
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Array-CGH: Limitations
Waves (GC)
Technical
Background
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Array-CGH: Limitations
Technical
Samples:
- Fresh: Blood, BM, other liquids
- Frozen tissues (cancer)
Quality
FFPE tissues
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Analysis
• DNA origin (% of tumoral cells, reactive cells)
• Balanced chromosomal aberrations: translocations, inversions
• Subclones, ploïdy
• Driver chromosomal aberrations Genomic databases
• Constitutional abnormalities (predisposition genes), mutations
• Chromosomal mechanism: FISH, karyotype
Array-CGH: Limitations
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FISH et ACPA : analyse comparative
FISH ACPA
Formation, GBEA et C.Q. Indispensable
Mise en œuvre +++
Tissus fixés +++ +/-
Analyse ciblée des C tumorales +++ -
Infiltrat tumoral 10% > 30 %
Automatisation +++ +/-
Coût / test + ++
Etude pangénomique - +++
Résolution + +++
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Clinical impact of Array-CGH analysis
Tumor classification
Prediction of patient outcome
Post surgical approach
1p / 19q co-deletionCDKN2A / CDKN2B deletion
10q loss / 7q gainPDGFRA or EGFR amplification
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• Diagnostic et pronostic individuel, choix du traitement
• ACPA :
Tissu frais, congelé ( FFPE samples)
Analyse pangénomique de haute résolution
• ACPA : limites
Taille du prélèvement, % de C tumorales, clones, ploïdie, hétérogénéïté
tumorale …
FISH en 2ème intention ou pour confirmation
Réseau (CHU Angers)
Facturation : RiHN / B
Conclusion
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Acknowledgments
Medical and Technical Teams:
Cytogenetic and Cellular Biology Department
Pathology Department
Clinical cancer center
PFGMC