Dec.18. 2016. Abbott user seminar Practical issues of FISH in solid tumors

1. Practical Issues of FISH Analysis
in Solid Tumors
Yosep Chong, M.D.
Department of Hospital Pathology,
Yeouido St. Mary’s Hospital,
College of Medicine, The Catholic University of Korea
ychong@catholic.ac.kr

2. Disclosure
This educational program is sponsored by Abbott Molecular, Korea.
The content of this presentation is consistent with all FDA requirements
when applicable and is the sole responsibility of Dr. Yosep Chong.
Speaker Honoraria : Abbott Molecular Korea

3. Content
1. General procedure of FISH test
2. Assessment of sample adequacy
1) Proper tissue fixation
2) Decalcified samples
3) DNA degradation in paraffin samples
4) Tumor cell adequacy
5) Sample type & characteristics
6) Strategies for increasing specimen availability
7) Selection of area of interest

4. Content
3. Quality control in Test procedure
1) Tissue section,
2) Baking & deparaffinization
3) Protease digestion
4) Denaturation
4. Quality control in Interpretation
1) Importance of Histology-FISH matching
2) Probe types and characteristics
3) Truncation artifact
4) Positive controls
5. Trouble shooting
6. External QC program
5) Hybridization
6) Washing
7) Mounting
8) Image acquisition or Scanning
.
5) Cutoffs
6) Other considerations

5. Introduction
Yeouido St. Mary’s Hospital FISH test
• 17 probes, 30 referral hospital laboratories
• Annually 3000 cases
• Average TAT 2.8 days
• BioView Autoscanning and Image analysis
• http://www.cmcsungmo.or.kr:2002/popup/Molecular_01.html
• TEL (+82)-02-3779-1298

6. General procedure of FISH test of solid tumors 1
2-4um Deparaffinization
Hemo-De
or Xylene x4
Tissue fixation
100% EtOH or
(degraded EtOH )
Dehydration
(or Rehydration)
Air Dry
Tumor
Marking by
pathologist
H&E slide
5 min
Section
marking
0.2N HCL 20 min
Unmasking
pH 6.0 Citric acid
80°C 30 min
or microwave
Pretreatment
Protease
25mg/1mL 20 min
Protease digestion
H2O
3 min
85°C x 20 min or 95°C x 10 min
Incubation at 37°C for probe
12~16 hrs
Co-denaturation
And Hybridization
2X SCC
3 min
H2O
1 min
2X SCC
5 min
x2
2X SCC
5 min
x2
Dry
5 min
Dry
5 min
Probe
Cover sliding and
Rubber cement
sealing
10% buffered
Formalin
Baking
56°C 2hr
Copyright © Yosep Chong
H&E scanning

7. Post hybridization washing
0.4X SCC/0.03% Tween20
or NP40 at 73°C 2 min
DAPI counterstaining
& cover sliding
Test info input
& Tissue matching
Remove cement sealing
and cover slides
2X SCC
3 min
2X SCC soaking
Dry
5 min
General procedure of FISH test of solid tumors 2
Copyright © Yosep Chong
2X SCC/0.01% Tween20
or NP40 at RT 1 min
DAPI 10uL
5X dry scan
H&E (or IHC)
scanning (opt)
10X dry scan
DAPI Image capture
or scanning
60X oil scan
Signal capture
or ScanningSignal enumeration
& interpretation
Preparing
report
FISH analysis report
ID OOO 50 / F
Breast cancer Bx
Test : HER2/CEP6
RESULT
Analysed cells :20
HER2:CEP6 raEo :1.0
NEGATIVE

8. Assessment of sample adequacy
Proper tissue fixation
• Check fixation status of submitted samples
• Check and reduce cold ischemic time
• Avoid overfixation

9. Archives of Pathology &
Laboratory Medicine:
November 2014, Vol.
138, No. 11, pp.
1520-1530

10. Assessment of sample adequacy
Decalcified specimen
• Check the need of molecular studies beforehand
• Consider use of EDTA instead of acid decalcification
(or 5% formic acid (methanoic acid) less than 24 hrs)

13. Assessment of sample adequacy
DNA degradation in paraffin samples

14. Assessment of sample adequacy
Tumor cell adequacy
• Collect adequate number of good quality
tumor cells for each FISH analysis
– HER2 20/40 cells, ALK 50/100 cells
– 1p19q 100 cells, Lymphoma 200 cells
• Ensure the quality of analyzed cells
– Viable tumor cells with good signal quality
/ no stromal or inflammatory cells
– No background signals/ no necrosis or fibrosis

15. Sample type and characteristics
Cell block Bronchoscopic biopsy
Needle biopsy Resection specimen

16. Assessment of sample adequacy
Strategies for increasing specimen availability
1) Separate biopsy pieces into additional paraffin
blocks
2) Implement pre-cutting protocols
3) Limit re-facing (reduce recutting steps)
4) Improve IHC & FISH ordering and working flow
5) Use additional cut for suboptimal size sample
6) Combine patient’s multiple specimens

17. Assessment of sample adequacy
Selection of area of interest
• Avoid necrosis/ squeezing
• Avoid fibrosis / mucin / anthracotic pigments
• Try to select area with distinctive histologic
pattern of tumor cells
• Avoid abundant inflammatory cells
• Consider specimen and probe types

18. Assessment of sample adequacy
Selection of area of interest – Necrosis

19. Assessment of sample adequacy
Selection of area of interest – Necrosis

20. Assessment of sample adequacy
Selection of area of interest – Squeezing

21. Assessment of sample adequacy
Selection of area of interest – Fibrosis

22. Assessment of sample adequacy
Selection of area of interest – Fibrosis

23. Assessment of sample adequacy
Selection of area of interest – Macrophages

24. Assessment of sample adequacy
Selection of area of interest – Mucin

25. Assessment of sample adequacy
Selection of area of interest – Anthracosis

26. QC in test procedure
Tissue section - Overlapping

27. QC in test procedure
Baking & deparaffinization

28. QC in test procedure
Protease digestion – poor digestion

29. QC in test procedure
Protease digestion – overdigestion

30. QC in test procedure
Protease digestion – overdigestion

31. QC in test procedure
Denaturation – “speckling”

32. QC in test procedure
Hybridization
Factors that influence hybridization
• Temp
• pH
• Concentration of monovalent cations
• Presence of organic solvent (formamide)
Parameters for optimal hybridization condition
• Probe length
• Probe concentration
• Dextran sulfate
• Base mismatch
• Use of single-stranded vs. double-stranded probes

33. QC in test procedure
Denaturation and Hybridization
Target probe Denatura(on Hybridiza(on
ALK BAP 73℃ 5min 37℃ 14hr-24hr
Her2 72℃ 5min 37℃ 14hr-18hr
1p/19q 73℃ 5min 37℃ 6hr-16hr
MET 73℃ 3min 37℃ 16hr-20hr
MYC 73℃ 5min 37℃ Overnight
Urovysion 76℃ 3min 39℃ 14hr - 18hr
IGH BAP 73℃ 5min 37℃ Overnight
EWSR1 BAP 73℃ 5min 37℃ 6hr-16hr
LSI DNA probe 73℃ 5min 37℃ 12hr-16hr

34. QC in test procedure
Washing

35. QC in test procedure
Image acquisition or Scanning
Factors to check
• Z-stacking technique
• Scanning position change
• Filters
• DAPI counterstaining
• Depletion of fluorescent signals
• Auto CCD adjustment

36. QC in test procedure
Image acquisition or Scanning

37. QC in test procedure
Image acquisition or Scanning

38. QC in test procedure
Image acquisition or Scanning-DAPI

39. QC in test procedure
Image acquisition or Scanning - DAPI

40. Quality control in Interpretation
• Histology-FISH matching
• Probe types and characteristics
• Truncation artifact
• Positive control
• Cutoffs
• Other consideration

41. Importance of histology-FISH matching

42. Importance of histology-FISH matching

43. Quality control in Interpretation
Probe types &
characteristics
• Single color
• Dual color
• Multi color
• Break apart
• Dual fusion
NL AbNL
Tri-color Dual Fusion
NL AbNL
Dual color Dual Fusion
NL AbNL
Dual color Break apart

45. QC in Interpretation
Probe types and characteristics

46. QC in Interpretation
Truncation artifact

47. • Use widely accepted cutoff standards
– ASCO/CAP guideline for HER2
– EORTC or RTOG guideline for 1p19q
• Prepare relevant references
• Set up internal cutoffs using wild type
samples (e. g. probes for lymphomas)
• Update and correlate with clinical data
QC in Interpretation
Cutoffs

48. • Set up positive controls using known
positive samples or cell line
• Run a positive control along with the test
• Check the whole procedure regularly using
positive control
• Use internal control
• Optimize the test procedure using positive
controls
QC in Interpretation
Positive Controls

49. QC in Interpretation
Other considerations
ALK

50. ALK
QC in Interpretation
Other considerations

51. ALK
QC in Interpretation
Other considerations
ALK

52. ALK
QC in Interpretation
Other considerations

53. ALK
QC in Interpretation
Other considerations

54. Trouble shooting

55. Trouble shooting

56. Trouble shooting

58. Trouble shooting

59. External QC program
• 한국유전자검사평가원
– 연 2회 ( http://www.kigte.org )
• UK NEQAS ICC & ISH QC program
– 연 4회 ( http://www.ukneqasiccish.org )
• ASoC QAP
– ( https://www.hgsa.org.au/resources/quality-
assurance-programs )
• Interlaboratory comparative tests

60. REFERENCES
• Lisa Duffy, Liangtao Zhang, Donald R. Love and Alice M. George (2012). Quality Control
Considerations for Fluorescence In Situ Hybridisation of Paraffin-Embedded Pathology
Specimens in a Diagnostic Laboratory Environment, Latest Research into Quality Control,
Dr. Isin Akyar (Ed.), InTech, DOI: 10.5772/51266.
• James Cook, Fluorescence in situ hybridization (chapter 11), Cell and Tissue Based
Molecular Pathology, 1st Ed., Elsevier, 2009 PP.104-113.
• Lecture of Dr. Marileila Varella Garcia, FISHing to Improve Lung Cancer Survival and
Challenges and Opportunities in FISH testing. 16 May 2016, Abbott seminar, Korea
• B. Paige Bass, Kelly B. Engel, Sarah R. Greytak, and Helen M. Moore (2014) A Review of
Preanalytical Factors Affecting Molecular, Protein, and Morphological Analysis of Formalin-
Fixed, Paraffin-Embedded (FFPE) Tissue: How Well Do You Know Your FFPE Specimen?.
Archives of Pathology & Laboratory Medicine: November 2014, Vol. 138, No. 11, pp.
1520-1530
• Eugenia Haralambieva, Karin Kleiverda, David Y Mason, Ed Schuuring and Philip M Kluin,
Detection of three common translocation breakpoints in non-Hodgkin’s lymphomas by
fluorescence in situ hybridization on routine paraffin-embedded tissue Sections. J Pathol
2002; 198: 163–170.