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K Number
K100015
Device Name
VYSIS CLL FISH PROBE KIT (VYSIS LSI TP53 SPECTRUMORANGE/ATM SPECTRUMGREEN) AND LSI D135319 SPECTRUMORANGE/13Q34
Manufacturer
ABBOTT MOLECULAR, INC.
Date Cleared
2011-08-09

(582 days)

Product Code
OVQDAT
Regulation Number
866.6040
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP Authorized
Intended Use
The Vysis CLL FISH Probe Kit is intended to detect deletion of the LS1 TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence in peripheral blood specimens from untreated patients with B-cell chronic lymphocytic leukemia (CLL). The assay may be used to dichotomize CLL (the 13q-, +12, or normal genotype group versus the 11q- or 17p- group) and may be used as an aid in determining disease prognosis in combination with additional biomarkers, morphology, and other clinical information. The Vysis CLL FISH Probe Kit is not intended for use in selection of therapy or in monitoring of residual disease.
Device Description
The Vysis CLL FISH Probe Kit uses fluorescence in situ hybridization (FISH) DNA probe technology to determine deletions of the locus-specific identifier (LSI) TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence. The Vysis CLL FISH Probe Kit (List No. 4N02-020) consists of two DNA FISH probe sets and three general purpose reagents sufficient to process 20 assays. - . LSI TP53 SpectrumOrange/ATM SpectrumGreen Probe - LSI D13S319 SpectrumOrange/13q34 SpectrumAqua/CEP 12 SpectrumGreen Probe . - DAPI II Counterstain . - NP-40 . - 20X SSC Salt .
More Information

Agendia BV MammaPrint® (K062694)

Not Found

No
The device description and performance studies focus on traditional FISH technology and statistical analysis of signal patterns, with no mention of AI or ML.

No.

The device is specified for diagnosis and prognosis, not for treating or preventing a disease.

Yes

The device is intended to detect deletion or gain of specific gene sequences in peripheral blood specimens from untreated patients with CLL, and "may be used as an aid in determining disease prognosis." This falls under the definition of a diagnostic device as it provides information for the diagnosis and prognosis of a disease.

No

The device is a kit containing DNA FISH probes and general purpose reagents, which are physical components used in a laboratory assay. It is not solely software.

Based on the provided information, the Vysis CLL FISH Probe Kit is an IVD (In Vitro Diagnostic).

Here's why:

  • Intended Use: The intended use explicitly states that the device is "intended to detect deletion... and gain... in peripheral blood specimens from untreated patients with B-cell chronic lymphocytic leukemia (CLL)." It also mentions its use as an aid in determining disease prognosis. This clearly indicates that the device is used to examine specimens derived from the human body to provide information for diagnostic purposes.
  • Device Description: The description details a kit containing probes and reagents used to perform a test on biological samples (peripheral blood).
  • Anatomical Site: The device is used on "Peripheral blood specimens," which are human biological samples.
  • Performance Studies: The document describes various analytical performance studies (Specificity, Sensitivity, Precision, Reproducibility, Method Concordance) conducted on human specimens to demonstrate the device's ability to accurately detect the specified genetic abnormalities.
  • Clinical Utility: The mention of supporting published clinical studies demonstrating correlation with overall survival in CLL further reinforces its diagnostic purpose.

The definition of an In Vitro Diagnostic (IVD) device is a medical device that is used to perform tests on specimens taken from the human body, such as blood, urine, or tissue, to detect diseases, conditions, or infections. The Vysis CLL FISH Probe Kit fits this definition perfectly.

N/A

Intended Use / Indications for Use

The Vysis CLL FISH Probe Kit is intended to detect deletion of the LS1 TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence in peripheral blood specimens from untreated patients with B-cell chronic lymphocytic leukemia (CLL). The assay may be used to dichotomize CLL (the 13q-, +12, or normal genotype group versus the 11q- or 17p- group) and may be used as an aid in determining disease prognosis in combination with additional biomarkers, morphology, and other clinical information. The Vysis CLL FISH Probe Kit is not intended for use in use in selection of therapy or in monitoring of residual disease.

Product codes (comma separated list FDA assigned to the subject device)

OVQ

Device Description

The Vysis CLL FISH Probe Kit uses fluorescence in situ hybridization (FISH) DNA probe technology to determine deletions of the locus-specific identifier (LSI) TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence.

The Vysis CLL FISH Probe Kit (List No. 4N02-020) consists of two DNA FISH probe sets and three general purpose reagents sufficient to process 20 assays.

  • LSI TP53 SpectrumOrange/ATM SpectrumGreen Probe
  • LSI D13S319 SpectrumOrange/13q34 SpectrumAqua/CEP 12 SpectrumGreen Probe
  • DAPI II Counterstain
  • NP-40
  • 20X SSC Salt

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Fluorescence microscopy

Anatomical Site

Peripheral blood

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Nonclinical Studies

Analytical Specificity

  • Study Type: Visual demonstration of probe hybridization specificity.
  • Sample Size: 5 karyotypically normal males (metaphase chromosomes from peripheral blood cultures). 20 metaphase nuclei evaluated per slide, total 200 target loci.
  • Key Results: The analytical specificity for each probe (Vysis LSI TP53 SpectrumOrange, Vysis LSI ATM SpectrumGreen, Vysis LSI D13S319 SpectrumOrange, Vysis LSI 13q34 SpectrumAqua, Vysis CEP 12 SpectrumGreen) was 100% (200/200).

Analytical Sensitivity

  • Study Type: Evaluation of percentage of scoreable interphase nuclei with expected normal signal pattern.
  • Sample Size: 25 karyotypically normal patients (interphase nuclei from peripheral blood cultures). 200 nuclei evaluated per specimen (100 nuclei by each of two technologists), total 5000 scoreable nuclei.
  • Key Results:
    • LSI TP53 SpectrumOrange: 97.98% (4899/5000)
    • LSI ATM SpectrumGreen: 98.68% (4934/5000)
    • LSI D13S319 SpectrumOrange: 98.60% (4930/5000)
    • CEP 12 SpectrumGreen: 98.94% (4947/5000)

Analytical Characterization of Normal Cut-off Values

  • Study Type: Determination of maximum percentage of abnormal signal patterns in normal specimens.
  • Sample Size: 25 karyotypically normal patients (interphase nuclei from peripheral blood cultures). 200 nuclei evaluated per specimen.
  • Key Results: Normal cut-off values were calculated using the beta inverse function.
    • Vysis LSI TP53 SpectrumOrange (1 signal): 14 out of 200 nuclei (7.0%)
    • Vysis LSI ATM SpectrumGreen (1 signal): 12 out of 200 nuclei (6.0%)
    • Vysis LSI D13S319 SpectrumOrange (1 signal): 11 out of 200 nuclei (5.5%)
    • Vysis LSI D13S319 SpectrumOrange (0 signal): 3 out of 200 nuclei (1.5%)
    • Vysis CEP 12 SpectrumGreen (3 signals): 5 out of 200 nuclei (2.5%)

Precision

  • Study Type: Precision analysis of percentages of abnormal cells.
  • Sample Size:
    • Precision Study 1: 10-member blinded slide panel (2 negative, 8 with at least one abnormality).
    • Precision Study 2: 8 different patient specimens.
  • Description: Studies involved testing three lots of probes on two/three days. For each specimen, 200 nuclei were evaluated.
  • Key Results: Tables 4-8 show mean and standard deviations of observed percentages of abnormal cells for negative, positive, and specimens near cut-off for each probe. Precision was analyzed separately for each specimen per probe.

Reproducibility

  • Study Type: Inter-laboratory reproducibility study.
  • Sample Size: 20-member blinded slide panel representing five Dohner classifications (13q-, No cytogenetic abnormality, +12, 11q-, 17p-).
  • Description: Three individual laboratories tested the panel with 10 slides per day for two days, using the same lots of probes.
  • Key Results:
    • Overall agreement between sites by probe (Table 9):
      • TP53 (17p-): 100% (20/20 agree)
      • ATM (11q-): 90% (18/20 agree)
      • CEP 12 (+12): 100% (20/20 agree)
      • D13S319 1x (13q-): 90% (18/20 agree)
      • D13S319 2x (13q-): 90% (18/20 agree)
    • Reproducibility based on Generalized Kappa Statistic for Prognostic Category (Table 10): Kappa = 0.86, indicating "Almost Perfect" agreement.
    • Reproducibility by Site based on Fisher's Exact Test (Table 11): p-value 0.8396.
    • Reproducibility by Probe based on Fisher's Exact Test (Table 12): p-values for probes range from 0.7495 to 1.000.

Clinical Utility

  • Study Type: Literature review supporting the clinical utility of FISH for CLL prognosis.
  • Key Results:
    • References to studies by Döhner et al. and Shanafelt, et al., showing correlation between genomic alterations detected by FISH and disease progression/overall survival in CLL.
    • Shanafelt et al. study (151 patients) established correlation between overall survival and FISH risk category for CLL at diagnosis, dividing patients into good/intermediate and poor prognosis groups.
    • The clinical utility of the Vysis CLL FISH Probe Kit is supported by its high concordance with the assay used in the Shanafelt study.
    • Mentions Tam et al. study on 17p- CLL patients and NCCN CLL practice guideline.

Method Concordance

  • Study Type: Clinical validity study demonstrating concordance to a Reference FISH Test (RFT).
  • Sample Size: 64 specimens whose Döhner classifications were based on previous RFT results.
  • Data Source: Specimens de-identified and randomized for testing with AMT (Abbott Molecular Test - Vysis CLL FISH Probe Kit) and RFT.
  • Annotation Protocol: A Dohner classification was assigned to each AMT and RFT result, and the Prognostic Category was determined.
  • Key Results: The percent agreement between the AMT and RFT for Prognostic Category was 97% (62/64) with a lower bound of the one-sided 95% confidence interval of 90%.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

See "Summary of Performance Studies" for sensitivity, specificity, and agreement data.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Agendia BV MammaPrint® (K062694)

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

§ 866.6040 Gene expression profiling test system for breast cancer prognosis.

(a)
Identification. A gene expression profiling test system for breast cancer prognosis is a device that measures the ribonucleic acid (RNA) expression level of multiple genes and combines this information to yield a signature (pattern or classifier or index) to aid in prognosis of previously diagnosed breast cancer.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Gene Expression Profiling Test System for Breast Cancer Prognosis.” See § 866.1(e) for the availability of this guidance document.

0

510(k) Summary

510(k) Number: K100015

Device Name: Vysis CLL FISH Probe Kit

Purpose of the Submission

The purpose of this 510(k) is to gain clearance to market the Vysis CLL FISH Probe Kit (List No. 4N02-020).

Official Correspondent to the File

Name:Dr. Nancy Bengtson
Title:Manager, Regulatory Affairs
Telephone:(224) 361-7087
Fax:(847) 775-6777
Email:Nancy.Bengtson@abbott.com
Name:Ms. Pamela L. Swatkowski
Title:Director of Regulatory Affairs
Telephone:(224) 361-7013
Fax:(847) 775-6777
Email:Pamela.Swatkowski@abbott.com
Address:Abbott Molecular Inc.
1300 E. Touhy Avenue
Des Plaines. IL 60018

Date of Preparation

August 9, 2011

Manufacturer

Abbott Molecular Inc. is the legal manufacturer of the Vysis CLL FISH Probe Kit (List

No. 4N02-020).

Name:Timothy Zurow, PhD
Title:Director Manufacturing Operations
Telephone:(224) 361-7379
Fax:(224) 361-7438
Email:timothy.zurow@abbott.com

Vysis CLL FISH Probe Kit 510(k) K100015 August 9, 2011

510(k) Summary Page 1 of 20

1

| Address: | Abbott Molecular Inc.
1300 E. Touhy Avenue
Des Plaines, IL 60018 |

----------------------------------------------------------------------------------

Establishment Registration No.: 3005248192

Intended Use

The Vysis CLL FISH Probe Kit is intended to detect deletion of the LS1 TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence in peripheral blood specimens from untreated patients with B-cell chronic lymphocytic leukemia (CLL). The assay may be used to dichotomize CLL (the 13q-, +12, or normal genotype group versus the 11q- or 17p- group) and may be used as an aid in determining disease prognosis in combination with additional biomarkers, morphology, and other clinical information. The Vysis CLL FISH Probe Kit is not intended for use in selection of therapy or in monitoring of residual disease.

Trade Name

Vysis CLL FISH Probe Kit

Common Name

Fluorescence In Situ Hybridization (FISH) reagents

Classification

Class II

Regulation Number

21 CFR 866.6040 Gene expression profiling test system for breast cancer prognosis

Product Code

OVQ, Chronic Lymphocytic Leukemia FISH Probe Kit

Predicate Devices

Agendia BV MammaPrint® (K062694)

Vysis CLL FISH Probe Kit 510(k) K100015 August 9, 2011

510(k) Summary Page 2 of 20

2

Comparison with Predicate

Not applicable. Clearance is supported by published clinical study and method concordance bridging studies.

Device Description

The Vysis CLL FISH Probe Kit uses fluorescence in situ hybridization (FISH) DNA probe technology to determine deletions of the locus-specific identifier (LSI) TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence.

The Vysis CLL FISH Probe Kit (List No. 4N02-020) consists of two DNA FISH probe sets and three general purpose reagents sufficient to process 20 assays.

  • . LSI TP53 SpectrumOrange/ATM SpectrumGreen Probe
  • LSI D13S319 SpectrumOrange/13q34 SpectrumAqua/CEP 12 SpectrumGreen Probe .
  • DAPI II Counterstain .
  • NP-40 .
  • 20X SSC Salt .

Background on Chronic Lymphocytic Leukemia (CLL)

Currently, most patients diagnosed with CLL have early-stage disease (Rai stage 0 or 1). Patients with early-stage CLL are a heterogeneous group; approximately 30% to 50% are at high risk of accelerated disease progression, and the remainder may live for decades and possibly never require therapy. Recent insights into the biological characteristics of leukemic B cells have led to the discovery of new prognostic tools (immunoglobulin variable-region heavy chain gene mutation status, cytogenetic abnormalities assessed by FISH, and Z-chain-associated protein kinase-70 protein expression) that can contribute to the identification of patients with early-stage disease who are at high risk for early disease progression. '

Routine karyotype analysis only detects chromosomal aberrations associated with CLL in 40% to 50% of the cases. Use of FISH and other technologies have detected genomic

510(k) Summary Page 3 of 20

3

abnormalities in over 80% of cases of CLL. The common genomic aberrations seen are trisomy 12 and deletions of 13q, 17p, and 11q.2-4

Several published studies suggest that some of these chromosomal abnormalities may be correlated with various disease parameters.5-8

The Vysis CLL FISH Probe Kit uses FISH DNA probe technology to determine deletion status of probe targets for LSI TP53 (containing tumor protein p53 gene, located on chromosome 17p), LSI ATM (containing ataxia telangjectasia mutated gene, located on chromosome 11q), and LSI D13S319 (containing marker D13S319, located on chromosome 13q), as well as determining trisomy 12 with CEP12 (D12Z3 alpha satellitelocation chromosome 12).

The Vysis CLL FISH Probe Kit includes LSI 13q34 (containing lysosomal-associated membrane protein I gene, located on chromosome 13g) as a quality control probe.

Technological Description of the Device

FISH is a technique that allows visualization of specific nucleic acid sequences within a cellular preparation. Specifically, FISH involves precise annealing of a single-stranded, fluorophore-labeled DNA probe to a complementary target sequence. Hybridization of the probe with the cellular DNA site is visible by direct detection using fluorescence microscopy. Interpretation of FISH results should be made utilizing appropriate controls and analytical techniques as well as taking into consideration other clinical and diagnostic test data.9

Peripheral blood cells from CLL patients are attached to microscope slides using standard cytogenetic procedures. The resulting specimen DNA is denatured to single-stranded form and subsequently allowed to hybridize with the probes of the CLL FISH Probe Kit. Following hybridization, the unbound probe is removed by a series of washes, and the nuclei are counter-stained with DAPI, a DNA-specific stain that fluoresces blue, Hybridization of the Vysis LSI TP53 SpectrumOrange, LSI ATM SpectrumGreen, LSI D13S319 SpectrumOrange, LSI 13q34 SpectrumAqua, and CEP 12 SpectrumGreen probes is viewed using a fluorescence microscope equipped with appropriate excitation

Vysis CLL FISH Probe Kit 510(k) K100015 August 9, 2011

510(k) Summary Page 4 of 20

4

and emission filters, allowing visualization of the orange, green, and aqua fluorescent signals.

In a cell with normal copy numbers of the LSI TP53 SpectrumOrange and LSI ATM SpectrumGreen probe targets, two orange and two green signals will be expected. In a cell with normal copy numbers of Vysis LSI D13S319 SpectrumOrange and CEP 12 SpectrumGreen probe targets, two orange signals and two green signals will be expected. Enumeration of the Vysis LSI TP53 SpectrumOrange, LSI ATM SpectrumGreen, LSI D13S319 SpectrumOrange, and CEP12 SpectrumGreen signals provides a mechanism for determining absolute copy number of the probe targets and the presence of the aberrations of interest. Any aberrations detected are used to determine Döhner Classification and prognostic category.

Summary of Nonclinical Studies

Analytical Specificity

Analytical specificity is defined as the percentage of signals that hybridize to the correct locus and no other location. This test is a visual demonstration that each probe hybridizes specifically to the expected chromosome location. The analytical specificity of the probes in the Vysis CLL FISH Probe Kit for their respective chromosome target loci was established using metaphase chromosomes prepared from peripheral blood cultures of five karyotypically normal males on microscope slides. The hybridization location of each FISH signal on chromosomes of 20 consecutive metaphase nuclei on each of 5 slides was evaluated by one technologist for a total of 200 target loci.

For each probe and sample, the number of metaphase chromosome FISH signals hybridized to the correct locus and the number of metaphase chromosome FISH signals hybridized to the incorrect locus were enumerated. The specificity of each probe was calculated as the number of metaphase chromosome FISH signals hybridized to the correct locus divided by the total number of metaphase chromosome FISH signals hybridized and multiplied by 100 to give a percentage. The analytical specificity for each probe of the Vysis CLL FISH Probe Kit was 100% (200/200) (Table 1).

5

| | | No. of Metaphase
Chromosome Signals | | | |
|----------------------------------------|----------------------------------|----------------------------------------------|--------------------------------|--------------------|-----------------------------------|
| Probe | Correct
Cytogenetic
Target | Hybridized to
the Correct
Target Locus | Total
Hybridized
Signals | Specificity
(%) | 95%
Confidence
Interval (%) |
| Vysis LSI TP53
SpectrumOrange | 17p13.1 | 200 | 200 | 100 | 98.17, 100 |
| Vysis LSI ATM
SpectrumGreen | 11q22.3 | 200 | 200 | 100 | 98.17, 100 |
| Vysis LSI
D13S319
SpectrumOrange | 13q14.3 | 200 | 200 | 100 | 98.17, 100 |
| Vysis LSI 13q34
SpectrumAqua | 13q34 | 200 | 200 | 100 | 98.17, 100 |
| Vysis CEP 12
SpectrumGreen | 12p11.1-q11 | 200 | 200 | 100 | 98.17, 100 |

Table 1. Analytical Specificity of Probes in Vysis CLL FISH Probe Kit

Analytical Sensitivity

Analytical sensitivity is defined as the percentage of scoreable interphase nuclei with the expected normal signal pattern. The expected normal interphase signal pattern for all probes in the Vysis CLL FISH Probe Kit is two signals per nucleus.

The analytical sensitivity of the probes in the Vysis CLL FISH Probe Kit for their respective chromosome target loci was established using interphase nuclei prepared from peripheral blood cultures of 25 karyotypically normal patients. For each specimen, the signal patterns of 200 nuclei were evaluated by counting the number of orange and green signals present for each probe target. Each technologist evaluated 100 nuclei per specimen for a total of 200 nuclei per specimen and 5000 scoreable nuclei from normal specimens.

The sensitivity (with 95% confidence intervals based on binomial distribution) was calculated as the percentage of scoreable interphase nuclei with the expected signal pattern of two signals per nucleus.

The Vysis CLL FISH Probe Kit has a sensitivity of 97.98% for the LSI TP53 SpectrumOrange probe, 98.68% for the LSI ATM SpectrumGreen probe, 98.60% for the LSI D13S319 SpectrumOrange probe, and 98.94% for the CEP 12 SpectrumGreen probe (Table 2).

510(k) Summary Page 6 of 20

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Number of Interphase NucleiSensitivity
ProbeWith Expected
Signal PatternScoreable
SignalsPercent (%)95%
Confidence
Interval
(%)
Vysis LSI TP53 SpectrumOrange4899500097.9897.55, 98.35
Vysis LSI ATM SpectrumGreen4934500098.6898.32, 98.98
Vysis LSI D13S319
SpectrumOrange4930500098.6098.23, 98.91
Vysis CEP 12 SpectrumGreen4947500098.9498.62, 99.21

Table 2. Analytical Sensitivity and Scoreable Percentage for Each Probe in the Vysis CLL FISH Probe Kit

Analytical Characterization of Normal Cut-off Values

The normal cut-off value, in association with FISH DNA probes, is defined as the maximum percentage of scoreable interphase nuclei with a specific abnormal signal pattern at which a specimen is considered normal for that signal pattern. The normal cutoff value is expressed in terms of a percentage or the actual number of abnormal nuclear FISH patterns per the standard number of nuclei tested. The criteria used to classify a nucleus as scoreable is located in the Interpretation and Result Reporting section of the package insert. The Quality Control and Signal Enumeration sections and the Dual Color Signal Counting Guide instruct the end user to determine whether an entire slide is adequate for signal enumeration and which types of cells/signals can be enumerated.

The normal cut-off values of the probes in the Vysis CLL FISH Probe Kit for their respective chromosome target loci were established using interphase nuclei prepared from peripheral blood cultures of 25 karyotypically normal patients. For each specimen, the signal patterns of 200 nuclei were evaluated by counting the number of orange and green signals present for each probe target.

The expected normal interphase signal pattern for all probes in the Vysis CLL FISH Probe Kit was two signals per nucleus. Since the specimen population does not fit a Gaussian distribution, the normal cut-off value was calculated using the beta inverse function. 9

7

The Vysis CLL FISH Probe Kit was shown to have normal cut-off values of 14 out of 200 nuclei evaluated (7.0%) for the LSI TP53 SpectrumOrange probe (1 signal), 12 out of 200 nuclei evaluated (6.0%) for the LSI ATM SpectrumGreen probe (1 signal), 11 out of 200 nuclei evaluated (5.5%) for the LSI D13S319 SpectrumOrange probe (1 signal), 3 out of 200 nuclei evaluated (1.5%) for the LSI D13S319 SpectrumOrange probe (0 signal), and 5 out of 200 nuclei evaluated (2.5%) for the CEP 12 SpectrumGreen probe (3 signals) (Table 3).

| Probe
(Abnormal Signal Pattern
of Interest) | Number of
Nuclei
Evaluated (n) | Maximum
Number of False-
Positive Patterns | Normal
Cut-off Value
(per 200 nuclei) | Normal Cut-
off Value
(%) |
|---------------------------------------------------|--------------------------------------|--------------------------------------------------|---------------------------------------------|---------------------------------|
| Vysis LSI TP53
SpectrumOrange (1 signal) | 200 | 8 | 14 | 7.0 (14/200) |
| Vysis LSI ATM
SpectrumGreen (1 signal) | 200 | 6 | 12 | 6.0 (12/200) |
| Vysis LSI D13S319
SpectrumOrange (1 signal) | 200 | 5 | 11 | 5.5 (11/200) |
| Vysis LSI D13S319
SpectrumOrange (0 signal) | 200 | 0 | 3 | 1.5 (3/200) |
| Vysis CEP 12
SpectrumGreen (3 signals) | 200 | 1 | 5 | 2.5 (5/200) |

Table 3. Analytical Characterization of Normal Cut-off Values

Precision

The precision of the probes in the Vysis CLL FISH Probe Kit was established using interphase nuclei prepared from two separate peripheral blood specimens lacking del(17p13.1), del(11q22.3), del(13q14.3), and trisomy 12, and eight additional specimens of which at least two specimens had one of the listed abnormalities (Precision Study 1).

This blinded 10-member slide panel consisting of both negative and positive specimens was used to test three lots of Vysis LSI TP53 SpectrumOrange/ATM SpectrumGreen and LSI D13S319 SpectrumOrange/13q34 SpectrumAqua/CEP 12 SpectrumGreen Probes on each of two days, and one of the three lots on a third day.

For each specimen, the FISH signal patterns of 200 nuclei were evaluated by counting the number of orange and green signals present for each probe target.

8

Precision was analyzed separately for each specimen per probe using the percentage of abnormal cells with the signal pattern(s) of interest.

A blinded panel created from eight different patient specimens was tested using three different DNA FISH probe lots on three days (the days were not required to be consecutive) (Precision Study 2). Precision analyses to challenge the normal cut-off values were performed using the percentage of cells with the signal pattern of interest and were analyzed separately for each panel member.

Precision Study 1 and Precision Study 2 mean and standard deviations of the observed percentages of abnormal cells of the negative, positive, and specimens near the normal cut-off are shown in Tables 4-8.

| Study | Sample | Category | n | Mean | Between-Day
(Within Lot)
Component SD | Between-Lot
Component
SD | Total SDb |
|-------|--------|-----------|---|------|---------------------------------------------|--------------------------------|-----------|
| 1 | 1 | Negative | 7 | 2.6 | 1.23 | 0.00 | 1.23 |
| 1 | 2 | Negative | 7 | 2.9 | 1.58 | 1.11 | 1.94 |
| 1 | 3 | Negative | 7 | 3.8 | 3.48 | 0.00 | 3.48 |
| 1 | 4 | Negative | 7 | 3.1 | 2.00 | 0.00 | 2.00 |
| 1 | 5 | Negative | 7 | 2.7 | 0.76 | 0.65 | 1.00 |
| 1 | 6 | Negative | 7 | 2.4 | 2.90 | 0.00 | 2.90 |
| 1 | 7 | Negative | 7 | 2.1 | 1.38 | 0.00 | 1.38 |
| 1 | 8 | Negative | 7 | 2.8 | 1.29 | 0.00 | 1.29 |
| 1 | 9 | Positive | 7 | 29.8 | 5.15 | 4.54 | 6.87 |
| 1 | 10 | Positive | 7 | 73.2 | 5.29 | 0.00 | 5.29 |
| 2 | 7 | Positivec | 9 | 13.6 | 3.11 | 0.00 | 3.11 |
| 2 | 8 | Positivec | 9 | 16.9 | 3.90 | 0.00 | 3.90 |

Table 4. Precision Analysis of Percentages of Abnormal Signal Patterns for Vysis LSI TP53 SpectrumOrange Probe [del(17q13.1(1 signal))]4

The mean and standard deviations are represented as percent abnormal signal patterns.

6 Total variance is the sum of the other variance components.

& Positive specimen near the normal cut-off.

9

| Study | Sample | Category | n | Mean | Between-Day
(Within Lot)
Component SD | Between-Lot
Component
SD | Total SDb |
|-------|--------|-----------|---|------|---------------------------------------------|--------------------------------|-----------|
| 1 | 1 | Negative | 7 | 1.6 | 0.54 | 0.59 | 0.80 |
| 1 | 2 | Negative | 7 | 1.8 | 0.59 | 0.00 | 0.59 |
| 1 | 3 | Negative | 7 | 2.6 | 0.81 | 1.54 | 1.74 |
| 1 | 4 | Negative | 7 | 2.1 | 2.25 | 1.08 | 2.50 |
| 1 | 5 | Negative | 7 | 2.9 | 1.71 | 1.64 | 2.37 |
| 1 | 6 | Negative | 7 | 1.1 | 1.34 | 0.00 | 1.34 |
| 1 | 7 | Positive | 7 | 64.1 | 3.18 | 0.00 | 3.18 |
| 1 | 8 | Positive | 7 | 13.7 | 4.31 | 5.10 | 6.68 |
| 1 | 9 | Negative | 7 | 1.6 | 0.99 | 0.00 | 0.99 |
| 1 | 10 | Negative | 7 | 1.5 | 1.51 | 0.00 | 1.51 |
| 2 | 1 | Positivec | 9 | 8.4 | 3.12 | 0.91 | 3.25 |
| 2 | 2 | Positivec | 9 | 7.8 | 3.00 | 1.89 | 3.55 |
| 2 | 8 | Positivec | 9 | 18.7 | 3.46 | 0.00 | 3.46 |

Table 5. Precision Analysis of Percentages of Abnormal Signal Patterns for Vysis LSI ATM SpectrumGreen Probe [del(11q22.3)(1 signal)]ª

" The mean and standard deviations are represented as percent abnormal signal patterns.

b Total variance is the sum of the other variance components.

° Positive specimen near the normal cut-off.

、 ·

10

| Study | Sample | Category | n | Mean | Between-Day
(Within Lot)
Component SD | Between-Lot
Component
SD | Total SDb |
|-------|--------|-----------|---|------|---------------------------------------------|--------------------------------|-----------|
| 1 | 1 | Negative | 7 | 3.5 | 1.12 | 1.10 | 1.57 |
| 1 | 2 | Negative | 7 | 2.2 | 1.69 | 0.00 | 1.69 |
| 1 | 3 | Positive | 7 | 66.1 | 6.13 | 0.00 | 6.13 |
| 1 | 4 | Positive | 7 | 20.1 | 4.88 | 0.00 | 4.88 |
| 1 | 5 | Negative | 7 | 0.6 | 0.83 | 0.00 | 0.83 |
| 1 | 6 | Negative | 7 | 0.4 | 0.64 | 0.00 | 0.64 |
| 1 | 7 | Positive | 7 | 84.9 | 4.14 | 0.00 | 4.14 |
| 1 | 8 | Negative | 7 | 0.6 | 0.97 | 0.00 | 0.97 |
| 1 | 9 | Negative | 7 | 2.3 | 2.08 | 0.00 | 2.08 |
| 1 | 10 | Positive | 7 | 53.6 | 8.57 | 0.00 | 8.57 |
| 2 | 5 | Positivec | 9 | 5.8 | 2.49 | 0.00 | 2.49 |
| 2 | 6 | Positivec | 9 | 14.9 | 2.46 | 1.15 | 2.72 |

Table 6. Precision Analysis of Percentages of Abnormal Signal Patterns for Vysis LSI D13S319 SpectrumOrange Probe [del(13q14.3) (1 signal)]ª

ª The mean and standard deviations are represented as percent abnormal signal patterns.

b Total variance is the sum of the other variance components.

" Positive specimen near the normal cut-off.

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| | | | | | Between-Day
(Within Lot)
Component SD | Between-Lot
Component
SD | |
|-------|--------|-----------|---|------|---------------------------------------------|--------------------------------|-----------|
| Study | Sample | Category | n | Mean | | | Total SDb |
| 1 | 1 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 2 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 3 | Negative | 7 | 0.2 | 0.21 | 0.38 | 0.44 |
| 1 | 4 | Negative | 7 | 0.1 | 0.40 | 0.00 | 0.40 |
| 1 | 5 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 6 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 7 | Negative | 7 | 0.2 | 0.32 | 0.00 | 0.32 |
| 1 | 8 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 9 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 10 | Positive | 7 | 18.4 | 6.32 | 0.87 | 6.38 |
| 2 | 8 | Positivec | 9 | 19.8 | 2.53 | 0.00 | 2.53 |

Table 7. Precision Analysis of Percentages of Abnormal Signal Patterns for Vysis LSI D13S319 SpectrumOrange Probe [del(13q14.3) (0 signals)]ª

  • The mean and standard deviations are represented as percent abnormal signal patterns.

b Total variance is the sum of the other variance components.

· Positive specimen near the normal cut-off.

12

| Study | Sample | Category | n | Mean | Between-Day
(Within Lot)
Component SD | Between-Lot
Component
SD | Total SDb |
|-------|--------|-----------|---|------|---------------------------------------------|--------------------------------|-----------|
| 1 | 1 | Negative | 7 | 0.2 | 0.42 | 0.00 | 0.42 |
| 1 | 2 | Negative | 7 | 0.2 | 0.60 | 0.00 | 0.60 |
| 1 | 3 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 4 | Negative | 7 | 0.1 | 0.40 | 0.00 | 0.40 |
| 1 | 5 | Positive | 7 | 21.6 | 6.07 | 0.00 | 6.07 |
| 1 | 6 | Positive | 7 | 70.7 | 4.06 | 1.07 | 4.19 |
| 1 | 7 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 8 | Positive | 7 | 67.3 | 4.84 | 0.00 | 4.84 |
| 1 | .9 | Negative | 7 | 0.0 | 0.00 | 0.00 | 0.00 |
| 1 | 10 | Negative | 7 | 0.1 | 0.20 | 0.00 | 0.20 |
| 2 | 3 | Positivec | 9 | 8.3 | 2.77 | 0.00 | 2.77 |
| 2 | 4 | Positivec | 9 | 3.6 | 3.05 | 0.00 | 3.05 |

Table 8. Precision Analysis of Percentages of Abnormal Signal Patterns for Vysis CEP 12 SpectrumGreen Probe {trisomy 12 (3 signals)]4

" The mean and standard deviations are represented as percent abnormal signal patterns.

6 Total variance is the sum of the other variance components.

  • Positive specimen near the normal cut-off.

Reproducibility

Three individual laboratories tested a blinded 20-member slide panel consisting of specimens representing each of the five Dohner classifications:

  • . 13q- (monosomy or nullisomy) as sole abnormality
  • No cytogenetic abnormality .
  • . +12 without 17p- or 11q-
  • . 11q- without 17p-
  • . 17p-

The analysis was conducted with 10 slides per day for two days for a total of six runs across three testing sites. The same lots of Vysis LSI TP53 SpectrumOrange/ATM SpectrumGreen and LSI D13S319 SpectrumOrange/13q34 SpectrumAqua/CEP 12

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SpectrumGreen Probes were tested at each laboratory. Results shown in Table 9 show an overall agreement with specimens representing each of the five Dohner classifications.

DisagreeᵃOverall
Probe (Abnormality)Site 1Site 2Site 3Number
AgreeᵃNumber
TestedPercent
Agreement
TP53 (17p-)0002020100
ATM (11q-)200182090
CEP 12 (+12)0002020100
D13S319 1x (13q-)200182090
D13S319 2x (13q-)011182090

Table 9. Overall Agreement, Site to Site by Probe

ª Disagree = Number of specimens for which one's site result did not agree with the other sites' results.

·

.

Agree = Number of specimens for which all 3 sites agreed on results.

14

The reproducibility of the Vysis CLL FISH Probe Kit for each Prognostic Category demonstrated almost perfect agreement'4 using a generalized kappa statistic among the three testing sites (Table 10).

Prognostic Category
SampleIntermediate/Good PrognosisaPoor Prognosisa
130
230
330
430
530
630
730
830
930
1030
1130
1230
1303
1412
1503
1612
1703
1803
1903
2003
Kappa0.86
StrengthAlmost Perfect14

Table 10. Vysis CLL FISH Probe Kit Reproducibility Based on Generalized Kappa Statistic

ª Number of sites based on Prognostic Category.

15

Additionally, the data were analyzed by site and probe using the Fisher's Exact Test (Tables 11 and 12).

SitePrognostic CategoryIntermediate/GoodaPoora
1128
2128
3146
p-value0.8396

Table 11. Vysis CLL FISH Probe Kit Reproducibility by Site Based on Fisher's Exact Test

ª Number of panel members based on Prognostic Category.

Table 12. Vysis CLL FISH Probe Kit Reproducibility by Probe Based on Fisher's
Exact Test
Probe (Abnormality)SiteAbnormality
DetectedNo
Abnormality
Detectedp -value
TP 53 (17p-)14161.0000
2416
3416
ATM (11q-)12180.7495
2416
3416
D13S319 1X (13q- [x1])16140.8396
2812
3812
D13S319 2x (13q- [x2])12181.000
2317
3317
CEP 12 (+12)15151.000
2515
3515

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Clinical Utility

The traditional Rai and Binet clinical CLL staging systems are based on disease burden and have been useful for assigning patients to groups having similar survival times. 1911 These systems, however, are not effective in predicting survival in early-stage disease when most CLL cases are diagnosed. This has led to development of newer molecular markers in an attempt to differentiate those patients who are prone to rapid progression from those who have indolent disease.

In a pivotal study conducted by Döhner et al, titled "Genomic Aberrations and Survival in Chronic Lymphocytic Leukemia," genomic alterations as determined by FISH were found to be predictive for disease progression and overall survival. Multiple studies support the conclusion of Dohner et al that loss of 11q markers predicts reduced survival times as compared to other Dohner groups as determined by FISH aberrations.68 Such studies have led to the inclusion of FISH testing in the National Comprehensive Cancer Network (NCCN) practice guidelines as a means to determine CLL prognosis. 12

In a 2006 prospective study of 151 patients by Shanafelt, et al. utilizing Vysis FISH probes, a correlation was established between overall survival and FISH risk category for CLL at diagnosis. Patients were divided into two prognostic groups. They were assigned to the good/intermediate FISH prognosis group if there were no chromosomal aberrations or if only 13q- and/or +12 aberrations were present. If a chromosomal aberration of 17por I I q- was present, the patient was placed in the poor FISH prognosis group. Poor vs. good/intermediate FISH (P=0.004), age at diagnosis (P=0.0006), and Rai stage (P=0.0026) were each significantly associated with overall survival from diagnosis in univariate analysis. When all factors were included in multivariable Cox regression model, each of three factors still remained significant: poor vs. good/intermediate FISH (P=0.00022), age at diagnosis (P=0.000024); and Rai stage (P=0.00012).

The clinical utility of the Vysis CLL FISH Probe Kit has been established primarily from its high concordance with the assay employed in the publication of Shanafelt et al.

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Also, as noted in the Shanafelt study, all patients with the 17p- abnormality had between 24-94% of cells with this abnormality. Therefore, the effect of 17p- at very low levels could not be determined. In a recent publication on untreated 17p- CLL patients, Tam et al reported a 3-year overall survival of 92% for patients with