Clinical Validation of Cytocell Pathology Probes Shivanand Richardson Specialized Technician Molecular Pathology Department of Pathology, University Medical Center Utrecht The Netherlands 5th annual Cytocell FISH User Group Meeting 2017
Clinical Validation of Cytocell Pathology Probes
Shivanand Richardson
Specialized Technician Molecular Pathology
Department of Pathology, University Medical Center Utrecht
The Netherlands
5th annual Cytocell FISH User Group Meeting 2017
Molecular Pathology UMC Utrecht
Disclosures
• Collaboration with Cytocell
Content
• FISH diagnostics at University Medical Center Utrecht
• Cytocell Project workflow at University Medical Center Utrecht
• CytoVision Scanning and Image Analysis
• Clinical Validation of Cytocell Pathology Probes
Cytocell – UMC Utrecht collaboration
Cytocell
• Designs and manufactures FISH probes
• Validate FISH probes on patient material
• Designs new probes for new diagnostic purposes
• Quality requirements (ISO certification) clinical evaluation
UMC Utrecht
• JCI Accredited institutePathology department ISO-norm 15189
• Experienced with FISH
• Cannot give away patient material
• Continuous improvement in FISH diagnostics
Collaboration essential for creating and validating new FISH probes
Pathology department of UMC Utrecht conducted clinical
validation of FISH probes (provided by Cytocell) on patient
material relevant for that probe.
With this collaboration Cytocell has a partner for probe validation
on patient material, and UMC Utrecht would be the first to have
new validated probes in a diagnostic setting.
Cytocell – UMC Utrecht collaboration
Molecular Pathology
UMC Utrecht
Mutation analysis
NGS, Sanger, HRM
TranslocationsFISH, RT-PCR
CNVCopy number variation
MLPA, FISH, SNP array
MethylationArray, MS-MLPA
MSIMicrosatellite instability
B- and T- cel clonality
HPVHuman papillomavirus
Molecular Pathology UMC Utrecht
Content
• FISH diagnostics at University Medical Center Utrecht
• Cytocell Project workflow at University Medical Center Utrecht
• CytoVision Scanning and Image Analysis
• Clinical Validation of Cytocell Pathology Probes
UMC Utrecht FISH workflow
FISH Request (LMS)
HE scoring (tumor area & percentage)
FISH slide in oven
FISH starts 14:00, pretreatment, area marking on glass and probe o/n
FISH stringent wash, DAPI + Cytovisionscanning
CytoVision pdf report in UDPS (KMBP)
(Uniform Decentralized System PALGA)
Autorisation in UDPS (pathologist)
Day 1
Day 2
CytoVision analysis of images (technician + KMBP)
Evt. Slide sectioning
FISH: • Monday – Thursday (entire workflow)
• Friday (image analysis + report)
FISH results:Within 2-3 daysMax. 1 week (repeat analysis)
Min. 40 FISH requests per week
Daily max. 28 FISH slides (routine diagnostics + Cytocell FISH validation)
UMC Utrecht FISHNormal Breakapart AmplificationDeletion
13-18-21 ALK BCL2 BCL6 CEP6 CHOP cMET cMYC b.a.
cMYCfusion
COL1A/PDGFRB
CCND1 ETV6 EWSR1 FGFR1 FGFR3 FKHR (FOXO1)
FUS HER2 MALT MAML2 MDM2 MUM1 MYB NMYC
PLAG-1 RET ROS1 SYT (SS18)
TFE3 USP6 X-Y-18 YWHAE
• 32 FISH Probes for diagnostic use• Various manufacturers (Cytocell, Vysis, Zytolight)• 1 FISH protocol (in-house method)
UMC Utrecht FISH amounts
UMC Utrecht
FISH diagnostics started 2008LeicaDM5500 manual microscoop
+LASX software
Leica DM6000 Scanner + CytoVision softwareFISH diagnostics since 2014Scanner Validated
on 75 FISH cases
RET Cytocell -Manual
Manual microscope vs Scanner
RET Cytocell -Scanner
100x 63x
Content
• FISH diagnostics at University Medical Center Utrecht
• Cytocell Project workflow at University Medical Center Utrecht
• CytoVision Scanning and Image Analysis
• Clinical Validation of Cytocell Pathology Probes
Phase 1
Phase 2
Cytocell Project
Cytocell
• Defines the list of probes and
references
UMC Utrecht
• reviews the list of probes
• discusses/agrees with Cytocell
• checks the availability of tissue
Cytocell probe and reference probe
were tested per patient simultaneously
Workflow Cytocell Project
Request• Receive FISH Probe request from Cytocell
Tissue
• Pathologist advices what type of tissue to use
• Tissues is requested and provided by Biobank Tissue Facility UMC Utrecht
FISH
• Slides sectioning, HE (tumor evaluation by pathologist) and FISH
• FISH is performed by trained technicians
Analysis
• Imaging with scan microscope (or by manual microscope)
• Image analysis and scoring detailed categories
• Report results
• Images and results are evaluated by Clinical Molecular Biologist in Pathology
Report
• Scoring results provided to Cytocell
• Digital Image are provided to Cytocell
• Clinical evaluation report
Workflow diagram
• FISH probe validation
• Use of FISH scanner
• Use of TMA
• Order reference probe• Register in probe database• Test on normal tissue (check if concordant to datasheet)• Determine tumor test tissue• Request test tissue• Slide Sectioning• Conduct FISH• Image analysis and report
Workflow diagram
• Probe validation
• Use of FISH scanner
• Use of TMA
• Marked FISH processed slide • Determine Tumor area and
percentage in HE• Determine scan method based
on tumor percentage, cell density and sample size
• Imaging (Manual, Semi-automatic or Automatic)
• Scoring / Results
Workflow diagram
• Probe validation
• Use of FISH scanner
• Use of TMA
Benefits:• Less probe needed• Specific tumor area• Image analysis with different probes in same region
• Request TMA• Tissue selection in UDPS• Slide sectioning for HE• Tumor area selection on HE• Submit marked slides and FFPE blocks• TMA is designed and made• TMA slide sectioning• Tumor evaluation in HE
TMA Colon/ Lung / Tonsil
TMA NSCLC cases
Content
• FISH diagnostics at University Medical Center Utrecht
• Cytocell Project workflow at University Medical Center Utrecht
• CytoVision Scanning and Image Analysis
• Clinical Validation of Cytocell Pathology Probes
Scanning preparation
• Marking of tumor area on FISH slide.
• Automatic scanning: Mandatory for
automatic region detection.
• Semi-automatic: Guidance for
manual region selection.
Case 1Case 1
Case 1 Case 1FISH
Case 1HE
Manual scoring with detailed categories
• Frequently > 40 categories• Extremely long Excel datasheets• Labor intensive• Error prone
CytoVision probe templates
• Standardized diagnostic scoring• +/- 8 categories• Cell monitoring (aberrant cells)
Break-apart spot counting configuration
+
+
Break- Apart FISH spot counting configuration
Case 1
Region Markup
Case 1
Case 1
Region Markup
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Region Markup
Case 1
Region Markup
Case 1
Region Markup
Scanning
Case 1
Case analysis
Case 1
Case analysis
Case 1
Case analysis
Case 1
Case analysis
Case 1
Case analysis
Case 1
Case analysis
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Case 1
Content
• FISH diagnostics at University Medical Center Utrecht
• Cytocell Project workflow at University Medical Center Utrecht
• CytoVision Scanning and Image Analysis
• Clinical Validation of Cytocell Pathology Probes
Phase 1
Cytocell Project
Frequency of genomic alterations in lung adenocarcinoma
ALK break-apart probe (LPS019)
• Intended purpose: To identify FISH rearrangements of ALK gene on chromosome 2 in patients with
Non-small-cell lung carcinoma.
• Test tissue: Lung – Primary and Metastatic tissue
• Aberrant cells: break – apart and single red
• ALK cut off criteria : <10% not translocated, 10%-15% equivocal (translocation unlikely), >15%
translocation
Round 1 Round 2
Reference method - Vysis ALK break-apart probe (06N38-020)
Source of material Archive slides TMA
Patients tissue N=2810 positive for translocation
N=22 1 positive for translocation
Control tissue N=8 N=5
Result Positive cases showed break-apart and single red signals
100% Concordant results between Cytocell probe and reference method
ALK Cytocell ALK Vysis
Phase 1- ALK
ROS1-GOPC (FIG) break-apart/deletion probe (LPS046)
• Intended purpose: To identify FISH rearrangements of ROS1 gene on chromosome 6 in patients
with Non-small-cell lung carcinoma.
• Test tissue: Lung – Primary and Metastatic tissue
• Aberrant cells: break – apart and single green
• ROS1-GOPC (FIG) cut off criteria : <10% not translocated, 10%-15% equivocal (translocation
unlikely), >15% translocation
Round 1 Round 2
Reference method - Kreatech ROS1 (6q22) break-apart probe (KBI-10752)
Source of material Archive slides TMA
Patients tissue N=264 positive for translocation
N=22 2 positive for translocation
Control tissue N=5 N=5
Result Positive cases showed break-apart and single green signals
3 cases discordant, increased background and weak signal with Kreatech probe
ROS1 Cytocell ROS1 Kreatech
Phase 1 – ROS1- GOPC (FIG)
RET break-apart probe (LPS045)
• Intended purpose: To identify FISH rearrangements of RET gene on chromosome 10 in patients with
Non-small-cell lung carcinoma.
• Test tissue: Lung – Primary and Metastatic tissue
• Aberrant cells: break – apart and single red
• RET cut off criteria : <10% not translocated, 10%-15% equivocal (translocation unlikely), >15%
translocation
Round 1 Round 2
Reference method - ZytoLight SPEC RET Dual Color break-apart probe (Z-2148-200)
Source of material Archive slides TMA
Patients tissue N=242 positive for translocation
N=22 0 positive for translocationRET translocations are very rare <2%
Control tissue N=5 N=5
Result Positive cases showed break-apart and single red signals
100% Concordant results between Cytocell probe and reference method
RET Cytocell RET ZytoLight
Phase 1 – RET
Phase 1 ALK / ROS1 / RET Probe Remarks
➢ Moderate – Good signal quality.
➢ Increased background caused by collagen in Lung tissue.
➢ Cellient (Cytological material) displayed increased background.
➢ Signal strength varies per case and per probe.
➢ ROS1 probe frequently display 1 signal pair per cell.
➢ Overall, the ALK, ROS1 and RET Cytocell probe works good enough for
diagnostic purposes, as compared to the reference probes.
Phase 2
Cytocell Project
• Intended purpose: To identify FISH rearrangements of TFE3 gene on the X chromosome in patients
with Renal Cell Carcinoma.
• Test tissue: Renal (kidney)
• Aberrant cells: break – apart
• TFE3 cut off criteria : ≥10% translocation
TFE3 break-apart probe (LPS051)
Round 1
Reference method ZytoLight SPEC TFE3 Dual Color break-apart probe (Z-2109-200)
Source of material Sectioned slides
Patients tissue N=25 1 positive for translocation(rare translocation)
Control tissue N=7 (tonsil)
Result 100% Concordant results between Cytocell probe and reference method
Aberrant signal pattern gender specific:
•Male (XY): break-apart single red and single green signal (no fusion pair)
•Female (XX): fusion pair + break-apart single red and single green signal
TFE3 Cytocell TFE3 ZytoLight
Phase 2 – TFE3
➢ All cases concordant.
➢ Moderate – Good signal quality.
➢ Interpretation in low quality tissue material is difficult.
➢ Signal strength varies per case.
➢ Overall, the Cytocell TFE3 (LPS051) probe works good enough
for diagnostic purposes, as compared to the ZytoLight probe.
Phase 2 – TFE3 probe remarks
• Intended purpose: To identify FISH rearrangements of FOXO1 gene on chromosome 13 in patients
with Alveolar Rhabdomyosarcoma .
• Test tissue: Sarcoma
• Aberrant cells: break – apart
• FOXO1 cut off criteria : ≥10% translocation
FOXO1(FKHR) break-apart probe (LPS049)
Round 1
Reference method Vysis FOXO1 break-apart probe (03N60-020)
Source of material Sectioned slides
Patients tissue N=22 3 positive for translocation
Control tissue N=7 (tonsil)
Result 100% Concordant results between Cytocell probe and reference method
FOXO1 Cytocell FOXO1 Vysis
Phase 2 – FOXO1
➢ All cases concordant.
➢ Moderate – Good signal quality.
➢ Interpretation in low quality tissue material is difficult.
➢ Signal strength varies per case.
➢ Overall, the Cytocell FOXO1 (LPS049) probe works good enough
for diagnostic purposes, as compared to the Vysis probe.
Phase 2 – FOXO1 probe remarks
• Intended purpose: To identify FISH rearrangements of FUS gene on chromosome 16 in patients with a
Soft Tissue Sarcoma.
• Test tissue: Sarcoma
• Aberrant cells: break – apart
• FUS cut off criteria : ≥10% translocation
FUS break-apart probe (LPS050)
Round 1
Reference method Vysis FUS break-apart probe (03N58-020)
Source of material Sectioned slides
Patients tissue N=28 13 positive for translocation
Control tissue N=7 (tonsil)
Result 1 case discordant, 7 cases not interpretable
FUS Cytocell FUS Vysis
Phase 2 – FUS
➢ Moderate signal quality
➢ Most cases concordant
➢ 27 cases concordant, 1 case discordant.
discordance: Cytocell: 6% break-apart - no translocation
Vysis: 45% break-apart- translocation
High background signals and weak FISH signal with both probes.
➢ 7 cases not interpretable: Cytocell: 7 cases
Vysis: 3 cases
➢ Interpretation in low quality tissue is more difficult using the Cytocell FUS break
apart probe, although the Vysis probe gives in some cases still a weak but
interpretable signal.
➢ If a sample shows too much background and weak fluorescent signal, do not try to
interpret the FISH signals -> Repeat analysis.
Phase 2 – FUS probe remarks
Take home message
FISH diagnostics can be fast, accurate and robust
Standardized protocols are essential for FISH diagnostics
High throughput & automated image analysis is validated
Critical clinical evaluation on tissue samples is necessary
Cytocell project Collaboration
Cytocell:
Kerry McKenna (Study Co-ordinator)
Darleen Welford (Director of Regulatory Compliance)
Steve Chatters (Senior Product Manager – Pathology)
Stewart Kennedy
TICO europe:
Marie-Christine Vierhout
UMCU, Pathology:
• Roel de Weger
• Stefan Willems
• Ton Peeters
• Petra van der Weide
• Kevin van der Ven
• Manon Huibers
• Tessa Vermeer
• Shivanand Richardson
Molecular Pathology UMC Utrecht