I Casi difficili

1. I Casi difficili Massimo Barberis Histopathology and Molecular Diagnostics Unit

3. 10 - 8- 2011 20 – 8 - 2012 4148 mutational analyses performed for patients seeking for biological therapy at EIO Egfr 1517 K-ras 1387 B-raf 392 C-kit 453 Altri 399 392 B-RAF suitablefor 311 casesregardindgptswith metastatic melanoma 9 Unsatisfactoryspecimens unsuitablefordiagmosis 2.9 % 8 cases in 2011 1 case in 2012 4148

4. Criticalphasesforcriticalspecimens • Tumorcell / stromal and inflammatorycells • Qualityoftumorcells • Typeofnucleic acid extraction • Primerselection • Extraction • Nucleic acid mustbeas pure aspossible. • Removeinhibitors, competing NA • Evaluateextraction and purificationsystems • foryieldefficency and consistency

5. Organic DNA extraction method Cells lysed with a detergent and mixed with phenol/chloroform Ultrahigh purity of the product Warning : salt concentration and pH dangerous organic solvents time consuming Solid phase extraction methods Silica base or fiber glass based technology ( chaotropic substances such as guanidine hydrochloride) FFPE Qiagen extraction kit Automated nucleic acid extraction

6. Let’s revise the lesson… Primers design For a good hybridization : At least 18-28 nucleotides in lenght Match Tm of the primers Keep C-G content between 50-60% Design primers that are not complementary Avoid palindromic sequences (to avoid secondary structures) Avoid all known SNPs ( check data bases *) Consider the most conserved region of the sequence of interest genome-www2.stanford.edu/cgi-bin/SGD/web-primer frodo.wi.mit.edu/cgi-bin/primer3

7. Problems that are encountered in “difficult cases”: Lack of PCR product Low yield of the desired product Presence of nonspecific background bands due to mispriming or misextension of the primers Formation of primer-dimer that competes for amplification with the desired product

8. Quality and quantity of DNA template Spectrophotometry, Fluorometry, Electrophoresis Blood : PCR products between 100-200 bp require 2-4 ng FFPE : 20-200 ng Try even with 5-10 ng

9. Take intoconsideration and optimize the following • PCR parameters , ifnecessary • Amountofstartingtemplate • Primerconcentration • Typeofenzyme and enzymeconcentration • Usemodifiedthermostable DNA polymerasesthat • provideautomatichot-startallowingtemplate DNA and • primerstobemixedtogether and held at a temperature • above the thresholdof non specificbindingofprimerto • templatebeforeamplification • Magnesiumionconcentration • Buffer composition and pH • Annealing temperature and numberofcycles

10. A special recipe for a hopeless case…. • Various addition or cosolvents such as dimethyl sulfoxide (DMSO), glycerol, • Bovine Serum Albumin can improve amplification: • Increase the specificity • < Decrease the amount of non specific products interfering • with sequencing • an old hen makes good broth • McPherson MJ et . PCR Basics from Background to Bench. Springer Verlag, 2000 • Innis MA et al. PCR Application. Protocols for Functional Genomics. Academic pPress • 1999

11. The first amplification failed, but we tried again and we got a result in sequencing ….. ?? Is the result clinically reasonable or may be attributable to specimen related problems , or to the platform or to the software ?? Re-evaluate the agarose or acrylamide gel, the quality score and the software indicators The quality of the sequence determined for the wild type control may be useful as a comparison in identifying problems

12. No recognizablesignal Signal loss afterbase-calling Unexpectedstops Look forcauses

13. Troubleshooting low-quality sequencing can be complex and may involve investigating multiple possible causes Poor template ( low concentration, poor preparation ) Inadequate purification of template prior to sequencing Poor primer and template annealing Contaminating sequences in reaction mix Low Tm, primer-dimer formation Reagents not performing as expected And many others

14. Mutational Analysis Workflow SAMPLE IDENTIFICATION (PATHOLOGIST) TUMORAL CELL THRESHOLD: MACRODISSECTION DNA EXTRACTION: CONTROL OF DNA YELD AND QUALITY PCR: PRIMER DESIGN AND AMPLIFICATION CONDITIONS PURIFICATION OF SEQUENCING PRODUCTS CYCLE SEQUENCING PCR PRODUCTS CLEAN UP GEL ELECTROPHORESIS (EXOSAP) CAPILLARY ELECTROPHORESIS DATA ANALYSIS

15. PCR Product Purification PCR Product Removal of PCR reaction “residuals” Primersto avoid multiple sequence dNTPs Salt to avoid inhibition of sequencing enzyme Purified Template

16. Purification Methods of PCR Products Exo I/ SAP Column/beads Diluition Gel Purification Sequencing Reaction Our Experience: we compared columns vs Exo-Sap and we’ve choosen Exo-sap becouse is: fast and easy cheap high throughput in microplate format Disadvantage: does not eliminate non specific PCR products

17. Sequencing Reaction Purification Methods Columns Ethanol precipitation Magnetic Beads Gel Filtration BigDye XTerminator kit Sample Preparation for Injection • Our Experience: • We’re using BigDye Xterminator after comparison with Colums with Ethanol purification. • BigDye Xterminator: • Simplifies workflow: • flexible in throughput and makes formamide needless • No beads removal prior injection • Removes “dye blobs” efficiently • No danger to loose pellet (as ethanol) • Increase signal intensity

18. To selectively amplify low levels of variant alleles COLD PCR consists of a particular cycling protocol in which a denaturation step at a lower temperature is applied

20. The problem of pigmentation Deparaffized, rehydrated sections 0.5% of potassium permanganate solution for 60’ Washing with distilled water 1% ossalic acid for 1’ Wash three times with distilled water Dissect the sections and insert the sample in a tube Proceed to extraction AFIP red book : < ossalic acid and potassium permanganate concentration > Incubation time

21. 11 cases of metastatic heavy pigmented poorly preserved samples of metastatic MM depigmented pigmented BRAF ex 15 Amplification (Sanger) Valuablesequence result in 10/11 Valuablesequence result in 7/11 PyroMarkQ96 9/11 Cobas 7/11

22. Molecular Diagnostics Unit