Clinical Validation of Cytocell Pathology Probes...fusion COL1A/P DGFRB CCND1 ETV6 EWSR1 FGFR1 FGFR3...

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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

Content

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)

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

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

• 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 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 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

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

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Region Markup

Scanning

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Case analysis

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Content

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.

• 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)

N=22 2 positive for translocation

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.

• 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)

N=22 0 positive for translocationRET translocations are very rare <2%

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.

➢ 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

• FOXO1 cut off criteria : ≥10% translocation

FOXO1(FKHR) break-apart probe (LPS049)

Round 1

Reference method Vysis FOXO1 break-apart probe (03N60-020)

Patients tissue N=22 3 positive for translocation

Result 100% Concordant results between Cytocell probe and reference method

FOXO1 Cytocell FOXO1 Vysis

Phase 2 – FOXO1

➢ All cases concordant.

➢ 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

• FUS cut off criteria : ≥10% translocation

FUS break-apart probe (LPS050)

Round 1

Reference method Vysis FUS break-apart probe (03N58-020)

Patients tissue N=28 13 positive for translocation

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

Clinical Validation of Cytocell Pathology Probes...fusion COL1A/P DGFRB CCND1 ETV6 EWSR1 FGFR1 FGFR3...

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