Molecular Tes,ng on Cytology Specimens
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1 Molecular Tes,ng on Cytology Specimens Sco6 Boerner MD FRCPC Medical Director & Head of Cytopathology University Health Network Associate Professor, University of Toronto UNIVERSITY of TORONTO
2 Disclosures! S,ll don t have any.
3 Learning Objec-ves At the comple,on of this session the par,cipants will have acquired an understanding of:! Suitability of cytologic samples for molecular tes,ng! The variety of cytologic substrates for molecular tes,ng! How different cytologic sampling techniques differ in their poten,al as substrates for molecular tes,ng! The impact of pre- analy,cal processing on suitability for molecular tes,ng! Limita,ons of cell block produc,on
4 Typical Statement About Cytology and Molecular American Society of Clinical Oncology provisional clinical opinion: epidermal growth factor receptor (EGFR) Muta,on tes,ng for pa,ents with advanced non- small- cell lung cancer considering first- line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol May 20;29(15):2121-7
5 Typical Statement About Cytology and Molecular Consensus for EGFR muta,on tes,ng in non- small cell lung cancer: results from a European workshop. J Thorac Oncol Oct;5(10):
6 Myths of Molecular Tes,ng! Cytologic samples are inadequate for molecular tes,ng why is this said?! Cytology results are untrustworthy! Cytology samples are too small! Cytology prepara,ons adversely alter the sample! Cytology sampling not representa,ve! Biased results due to lesion heterogeneity
7 Cytology and Quan,ty of Tissue
8 DNA Based Studies! Condi,ons for detec,on of muta,ons:! Quan,ty of DNA! Minimum quan,ty dependent on technique used! 1 to 50 ng DNA (167 to 8,350 intact diploid nuclei)! DNA fragment length! Most assays require 100 to 400 bp fragments! Frequency of mutant alleles! Minimum frequency dependent on technique used! 1-5% for amplifica,on refractory muta,on system (ARMS) PCR / pyrosequencing! 20-30% for direct sequencing
9 Myth FNA Collects Too Li6le Tissue Ideally, a sample should contain at least 200 to 400 tumour cells Consensus for EGFR muta,on tes,ng in non- small cell lung cancer: results from a European workshop. J Thorac Oncol Oct;5(10):
10 Quan,ty of Cytology Samples (Boerner Generaliza,ons)! Resec,on specimens >>> cytology samples! Effusion specimens may be an excep,on! Excisional biopsy >> cytology samples! Effusion specimens may be an excep,on! Core biopsy samples a toss- up! Depends on lesional characteris,cs! Some cores < cytology samples! Some cores > cytology samples! Some cores = cytology samples
11 Quan,ty of Cytology Samples (Boerner Generaliza,ons)! Depends on type of cytology sample! Brushings / Scrapings samples! Small quan,ty! Washings samples! Small to rarely moderate quan,ty
12 Quan,ty of Cytology Samples (Boerner Generaliza,ons)! Effusion samples! Exceedingly variable! Virtually none to massive quan,ty! FNA samples! Moderate to large! Exceedingly aspirator dependent
13 Myth FNA Collects Too Li6le Tissue! Why the Boerner literature contradic,on?! Literature statements mainly based on cell blocks! Cell block produc,on is a very ineffec3ve process! Best to assume you lose 50 to 85% of the sample! Cell suspension 90+% loss! Tissue fragments 50% loss, size dependent
14 How Many Cells Are Needed?! Not well studied! Literature! For EGFR with micro- dissec,on (cytology slides &/ or cell blocks) cells (J Thorac Oncol Nov;3(11): , Br J Cancer Jan 15;98(1):154-60, Am J Clin Pathol Sep;138(3):377-81)! Personal experience at UHN! EGFR muta,ons detected in cell blocks with cells
15 Substrates For Molecular Tes,ng! Cell blocks! Prepared slides! Residual fixed sample! Fresh sample! Really fresh! Hot off the press! Residual fresh! FTA Cards! That les over stuff going moldy in your frig
16 DNA Recovery (Fresh vs. CytoLyt vs. Formalin) Sample 1 Sample 2 UHN in- house data
17 DNA Recovery (Fresh vs. CytoLyt vs. Formalin vs. Time) Sample 1 Sample 2 UHN in- house data
18 DNA From Slides (Effects of Air- Drying vs Wet Fixa,on) Cancer Cytopathol Jul;121(7):344-53
19 DNA From Slides (Effects of Fixa,ve, Stain &Moun,ng Medium) Cancer Cytopathol Jul;121(7):344-53
20 DNA From Slides (DNA Fragment Length & Cytoprep) Cancer Cytopathol Jul;121(7):344-53
21 Muta,on Detec,on Equivalency Reliability of EGFR and KRAS muta,on analysis on fine- needle aspira,on washing in non- small cell lung cancer. Lung Cancer Apr;80(1):35-8
22 FTA Cards Cancer Cytopathol Jun 25;120(3):206-14
23 FTA Cards for EGFR Cancer Cytopathol Dec 25;118(6):450-6
24 Tumour Purity
25 Purity of Tumour! Tumour:Normal (T:N) ra,o! Depends on type of cytology sample! Brushings / Scrapings low T:N ra,o! Washings low T:N ra,o! Effusion exceedingly variable T:N ra,o! 0 to 1.0 (virtually all tumour)! FNA high T:N ra,o! FNA enriches for tumour and is depleted of stroma! Natural micro- dissec,on
26 Purity of Tumour! How to improve tumour purity! Get an FNA! Micro- dissec,on! Prepared slides! Cell blocks! At UHN we do not rou,nely micro- dissect cell blocks
27 My Preferences for Molecular Tes,ng! Each case requires its own judgement! General guidelines! Fresh sample first! Hot off the press! The old stuff in the frig! Residual alcohol fixed sample! Cell block material! Scrape a slide! The destruc,on of slide is the hold back
28 Fixa,ve for Cell Blocks for DNA Studies! 10% neutral buffered formalin! Pre- alcohol fixed + 10% NBF! Avoid exposure to fixa,ves containing! Picric acid (Bouin s)! Mercury (B5, Zenker s, Helly s, Ridley s)! Low ph fixa,ves! Phenols! Heavy metals (zinc)
29 UHN Cell Block Protocol
30 Does Molecular Tes,ng Work on Cytology EGFR muta,on tes,ng in lung cancer: a review of available methods and their use for analysis of tumour,ssue and cytology samples. J Clin Pathol Feb;66(2): 79-89
31 The World is Changing (But s,ll have a way to go) Molecular tes,ng guideline for selec,on of lung cancer pa,ents for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, Interna,onal Associa,on for the Study of Lung Cancer, and Associa,on for Molecular Pathology. J Thorac Oncol Jul;8(7):823-59
32 Thank you Sco6 Boerner MD FRCPC Medical Director & Head of Cytopathology University Health Network Associate Professor, University of Toronto UNIVERSITY of TORONTO
33 Which of the following statements regarding cytology samples and molecular tes,ng is true? 1. FNA samples are of low cellularity and diluted by abundant normal elements. 2. Cells scraped from previously prepared cytology slides are suitable for DNA tes,ng. 3. DNA recovery from fresh (unfixed) effusion samples is poor. 4. FFPE cell block is a be6er choice for DNA tes,ng than residual sample in alcohol.
34 Which of the following chemicals has the least impact on DNA recovery for muta,on tes,ng? 1. Formalin 2. Ethanol 3. Picric acid 4. Xylene based moun,ng media 5. Fixa,ves containing heavy metals
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