PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com

Introduction 1

3DHISTECH’s Pannoramic 250 FLASH II is a Whole Slide Scanner capable of

scanning brightfield (H&E) and fluorescent slides in a desktop configuration. The

scanning system is capable of walk-away automation for up to 250 slides as well as

continuous mode slide feeding. It has the ability to capture fluorescent signals from

9 independent FL channels and up to 29 different Z-layers with perfect co-

localization.

The Dark Field Preview feature detects fluorescently stained tissues on the slides to

speed up image acquisition by acquiring only areas containing the sample.

Whole slide imaging with Pannoramic 250 FLASH II

• CymoGenDx Four-Color del-TECT™ PTEN FISH probe offers the highest

accuracy, sensitivity, and specificity for detection of PTEN deletion in FFPE

prostate tissue sections.

• 3DHISTECH’s P250 FLASH II Whole Slide Scanner offers the ability to scan H&E

and FISH slides with excellent image registration.

• Utilization of CymoGenDx’s FISH probes with Pannoramic 250 FLASH II Whole

Slide Scanner and FISHQuant Software offers an integrated workflow within a

clinical diagnostic setting.

Conclusion The CymoGenDx PTEN four-color del-TECT™ probe coupled with 3DHISTECH’s

FISHQuant analysis software significantly facilitated the analysis of genotypic

heterogeneity and varying clonality within different foci of tumor in prostate core

needle biopsies via superior scoring algorithms and detailed automated imaging

analysis of the 3DHISTECH platform. Overall, the approach provided robust and

highly reproducible results coupled with a highly-efficient automated scanning

system to increase accuracy and laboratory workflow.

The CymoGenDx four-color PTEN del-TECT™ FISH assay reduced the frequency of

misinterpretation and improved both the quality and throughput of FISH analyses

using clinical samples as demonstrated with 3DHISTECH’s automated platform.

CymoGenDx's del-TECT PTEN FISH probe combined with the 3DHISTECH

microscopy platform was able to overcome truncation artifact (a non-controllable

feature that is present in FFPE tissue sections and prevalent in core needle

biopsies) while accurately quantifying all known PTEN aberrations. The

pathologist is now able to visualize, analyze, quantify, and appropriately classify

very specific PTEN deletions by simultaneous comparisons of the FISH and H&E

stains from one unified workstation. This allows for enhanced precision and a

highly efficient automated FISH workflow.

PerkinElmer is the exclusive distributor of 3DHISTECH products in the USA.

FISHQuant software for automated FISH analysis

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Novel Four-Color PTEN del-TECT™ FISH Probe With High-Throughput Automated Microscopy for Efficient FISH Workflow

Authors: Annamaria Csizmadia*, Ferenc Szipocs*, Janet Park-Bewsher**, Mohammed Harris***, Peter Hartmayer***, Thomas Moss ***

*3DHISTECH, Hungary; ** PerkinElmer, USA; ***CymoGenDx, USA

Quality control standards in clinical laboratories using routine fluorescent in-situ

hybridization (FISH) analysis of formalin-fixed paraffin-embedded (FFPE)

sections are crucial as more genomic biomarkers are used for predictive and

prognostic oncology. The evaluation of FISH signals in interphase nuclei of FFPE

sections is affected by truncation and overlapping of the nuclei due to varying

cell density, tissue architecture differences and histological forms.

To overcome this, we incorporated the use of CymoGenDx’s four-color deletion-

detection PTEN del-TECT™ FISH Probe with the Pannoramic 250 FLASH II Whole

Slide Scanner and FISHQuant analysis software from 3DHISTECH. Figure 2b. CymoGenDx del-TECT™ and truncation artifact. A: normal cell with all four signals intact. B: truncated cell in which a loss is depicted due to slicing of the tissue section. C: true hemizygous deletion in which the PTEN gene is deleted while the flanking probes are retained. D and E represent potential truncation artifact in a 2-color FISH probe, where a large distance between the centromere 10 probe and the PTEN gene results in an increased rate of truncation.

A B C

D E

2-color FISH: Truncation or true deletion?

True Hemizygous Deletion

Figure 1. The CymoGenDx del-TECT™ PTEN FISH probe: RED centromeric probe, ORANGE PTEN target/test probe, GREEN centromeric flanking WAPAL probe, AQUA telomeric flanking FAS probe.

The PTEN four-color FISH probe comprises a centromeric “chromosome

enumeration” probe, a PTEN-specific gene probe, and control flanking probes on

both sides of the target PTEN probe. The incorporation of flanking probes

facilitates the differentiation of apparent loss of the chromosomal region due to

truncation artifact as opposed to true interstitial chromosomal deletion of PTEN.

A minimum threshold for apparent deletion frequency is applied to address the

heterogeneous and homogenous nature of tumor cell histology.

2 Key Features

3 CymoGenDx Four-Color del-TECT™ FISH Probe

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Figure 5. PTEN deletion A: All signals, B: PTEN(gold)+DAPI, C: CC10(red)+DAPI, D: FAS(aqua)+DAPI, E: WAPAL(green)+DAPI

Figure 6. Data Visualization Table for aneuploidy of CC10

A

C

B

D E

PTEN Four-Color del-TECT™ Whole Slide Images

FISHQuant is a semi-automatic image analysis software tool allowing the

analysis of digital FISH slides. One can define the clinically relevant tissue areas

on the digital slide with one or more annotations. The application works on the

whole digital slide or on selected annotated areas. FISHQuant segments nuclei

and spots automatically and scores the resulting data. For PTEN deletion it

measures the deletion clusters for the 3 colors involved.

Figure 3. 3DHISTECH’s Pannoramic 250 FLASH II Whole Slide Scanner

The Extended Focus feature compensates for the relatively shallow depth of field

of the high numerical aperture objective used for acquisition by combining the

in-focus parts from a series of z-layers into a single composite image. This then

enables the use of automated algorithms to evaluate FISH probes in tissue and

cytology samples.

Flat Field Correction removes the tiling effect due to uneven illumination and

other artifacts. Superlative stitching algorithms allow for the creation of seamless

whole slide images that bring digital microscopy on par or better than

conventional microscopy.

FISHQuant also uses the control to check for the numerical deviation of the

chromosome 10 signals. These two measurements are run automatically and

displayed on a two-dimensional “HistoPlot”.

This proof of concept study, using prostate core needle biopsies, demonstrates

that the PTEN deletion FISH signals captured on a digital slide and overlaid in

brightfield and fluorescence modes can be subsequently analyzed using

automated algorithms.

Figure 7. Homozygous PTEN deletion vs. CC10 signal “HistoPlot”. Copy number alterations of chromosome 10 is depicted on the y-axis while PTEN deletion patterns are depicted on the x-axis. A homozygous PTEN deletion (loss of both PTEN alleles) is presented above.

Typical PTEN FISH Patterns, Classifying criteria Patterns Signals for

CC10 Signals for

WAPAL Signals for

PTEN Signals for

FAS

Diploid of chromosome 10 2 2 2 2 Gain of chromosome 10 >2 >2 >2 >2 Hemizygous PTEN deletion 2 2 1 2 Hemizygous PTEN and FAS deletion 2 2 1 1

Hemizygous PTEN and WAPAL deletion 2 1 1 2

Hemizygous PTEN WAPAL FAS deletion 2 1 1 1

One copy loss of chromosome 10 1 1 1 1

Homozygous PTEN deletion 2 2 0 2 Homozygous PTEN and FAS deletion 2 2 0 <2

Homozygous PTEN and WAPAL deletion 2 <2 0 2

Homozygous PTEN WAPAL FAS deletion 2 <2 0 <2 Figure 4. 3DHISTECH's scanner is able to localize the FISH slide to the respective H&E slide for ease of comparison. Figure 2a. Truncation artifact during the sectioning process.

References: Paper in preparation "Incorporation of flanking probes reduces truncation losses for fluorescence in situ hybridization analysis of recurrent genomic deletions in tumor sections"

Maisa Yoshimoto, Olga Ludkovski, Jennifer Good, Robert J. Gooding, Jean McGowan-Jordan, Alexander Boag, Andrew Evans, Ming-Sound Tsao, Paulo Nuin, Jeremy A. Squire

Real PTEN deletion with normal 10 chromosome number.