Vysis CLL FISH Probe Kit

For In Vitro Diagnostic Use 

The Vysis CLL FISH Probe Kit is a test to detect deletion of the LSI TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence probe target via fluorescence in situ hybridization (FISH) in peripheral blood specimens from patients with B-cell chronic lymphocytic leukemia (CLL).

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description
 

The Vysis CLL FISH Probe Kit is a test to detect deletion of the LSI TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence probe target via fluorescence in situ hybridization (FISH) in peripheral blood specimens from patients with B-cell chronic lymphocytic leukemia (CLL) to aid in identifying those patients eligible for treatment with Venclexta® (venetoclax).

Vysis CLL FISH Probe Kit Contents

This kit contains five reagents sufficient to process 20 assays.  

An assay is defined as one 22 mm x 22 mm Vysis LSI TP53 SpectrumOrange/ATM SpectrumGreen probe hybridization area and one 22 mm x 22 mm Vysis LSI D13S319 SpectrumOrange/13q34 SpectrumAqua/CEP 12 SpectrumGreen probe hybridization area.

Each kit includes:

  • Vysis LSI D13S319 SpectrumOrange/LSI 13q34 SpectrumAqua/CEP 12 SpectrumGreen Probes (1 vial, 200 µL/vial) 
  • Vysis LSI TP53 SpectrumOrange/LSI ATM SpectrumGreen Probes (1 vial, 200 µL/vial)
  • DAPI II Counterstain (1 vial, 600 µL/vial)
  • NP-40 (2 vials, 200 µL/vial)
  • 20X Standard Sodium Citrate (SSC) Salt (1 bottle, 66 g) 

Indications and Limitations of Use

Intended Use

The Vysis CLL FISH Probe Kit is a test to detect deletion of the LSI TP53, LSI ATM, and LSI D13S319 probe targets and gain of the D12Z3 sequence probe target via fluorescence in situ hybridization (FISH) in peripheral blood specimens from patients with B-cell chronic lymphocytic leukemia (CLL).

Indications of Use

In untreated patients, 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 test is indicated for detecting deletion of the LSI TP53 probe target (17p-) as an aid in identifying those patients with CLL for whom treatment with VENCLEXTA® (venetoclax) is indicated.

Vysis CLL FISH Probe Kit is not intended for monitoring of residual disease.

The test is for prescription use only.

Limitations of the Procedure

  • FISH is a multiple step methodology that requires specialized training in the selection of the appropriate reagents, specimen preparation, processing, preparation of the FISH slide, and interpretation of the results.
  • When used to select CLL patients for treatment with VENCLEXTA® (venetoclax), the test is validated for peripheral blood specimens anticoagulated with sodium heparin only.
  • The Vysis CLL FISH Probe Kit is intended to be used in combination with additional biomarkers, morphology, and other clinical information.
  • Other signal patterns may occur, and metaphase analysis may be helpful in characterization of such patterns.
  • If a specimen has a low level abnormal FISH pattern, use of the appropriate single pass filter(s) to confirm the pattern is recommended. Failure to follow this recommendation may result in inaccurate identification of signal patterns.

CAUTION: United States Federal law restricts this device to sale and distribution to or on the order of a physician or to a clinical laboratory; and use is restricted to, by, or on the order of a physician.

studies
 

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.1,2 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 molecular markers that 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.3 Multiple studies support the conclusion of Döhner et al. that loss of 17p and of 11q markers predicts reduced survival times as compared to other Döhner groups as determined by FISH aberrations.4,5,6

Such studies have led to the inclusion of FISH testing in the NCCN practice guidelines as a means to determine CLL prognosis. CLL prognostic categories according to the current NCCN guidelines divide patients into 3 categories – favorable, neutral and unfavorable.7

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.6 Patients were divided into 2 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 11q- 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).6

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 by Shanafelt et al.6

Also, as noted in the Shanafelt study, all patients with the 17p- abnormality had between 24 to 94% of cells with this abnormality.Therefore, the effect of 17p- at very low levels could not be determined. In a publication on untreated 17p- CLL patients, Tam et al reported a 3-year overall survival of 92% for patients with < 25% 17p-deleted nuclei, vs. 54% for patients with ≥ 25% 17p-deleted nuclei (P=0.007).8

Method Concordance

A study was done comparing the method used in the Vysis CLL FISH Probe Kit and the method used by Shanafelt et al.6 This study established the clinical validity of the Vysis CLL FISH Probe Kit by demonstrating concordance to the FISH method used in the Shanafelt study. The clinical validity of the Vysis CLL FISH Probe Kit is documented via peer-reviewed literature. 

The study analyzed 64 specimens whose Döhner classifications were based on previous results using the Shanafelt FISH method. Table 22 shows the distribution of specimens analyzed, by Döhner classification. 

Table 22: Number of Specimens Analyzed, According to Döhner Classification

DÖHNER CLASSIFICATION  NUMBERS OF SPECIMENS ANALYZED
13q- (monosomy or nullisomy) as sole abnormality 13
Normal (no cytogenetic abnormalities) 12
+12 without 11q- or 17p- 14
11q- without 17p- 18
17p- 7

 

A Döhner classification was assigned to each result (Table 6), and the Prognostic Category was determined (refer to Table 7). The percent agreement between the Vysis CLL FISH Probe Kit and Shanafelt FISH method for Prognostic Category was 97% (62/64) with a lower bound of the one-sided 95% confidence interval of 90%. Overall agreement was defined as the percentage of specimens that had the same Prognostic Category when tested using the Vysis CLL FISH Probe Kit and the Shanafelt FISH method (refer to Table 23). 

Table 23. Concordance for Prognostic CategoryA

  REFERENCE SHANAFELT FISH TEST
Vysis CLL Fish Probe Kit Good/Intermediate Poor Total
Good/Intermediate 38 1  39
Poor 1 24  25
Total 39 25 64

A The values in this table represent the number of specimens

Clinical Trial Information

The clinical trial was a multi-center study testing peripheral blood specimens from relapsed or refractory CLL subjects. The efficacy of VENCLEXTA® (venetoclax) (a BCL2 inhibitor) was investigated in a single-arm trial of previously treated CLL patients with 17p deletion (Study 1, M13-982 trial). Study 1 was performed with the Vysis CLL FISH Probe Kit on peripheral blood specimens from relapsed/refractory CLL patients. A total of 167 subjects were screened for 17p deletion in the Study 1 main cohort.

Of the 167 subjects screened, 106 were enrolled in Study 1. Demographic and disease characteristics of the study population for Study 1 are provided in Table 24.

Table 24. Demographic and Disease Characteristics in Study 1

CHARACTERISTICS
N=106
Age (Years)   
  Median (Range) 67 (37-83)
Race, n (%)   
  Asian 0 (0)
  Black 3 (2.8)
  White/UnknownA

103

(97.2)
Gender, n (%)   
  Male 69 (65.1)
  Female 37 (34.9)
ECOG, n (%)   
  0 42 (39.6)
  1 55 (51.9)
  2 9 (8.5)
Number of Prior Therapies   
  Median (Range) 2.5 (1-10)

A Race unknown for one patient

In this study, 17p deletion status was determined using the Vysis CLL FISH Probe Kit. Efficacy data from Study 1 are provided in Table 25. The primary efficacy endpoint was overall response rate (ORR) as assessed by an Independent Review Committee (IRC) using the International Workshop for Chronic Lymphocytic Leukemia (IWCLL) updated Nation Cancer Institute-sponsored Working Group (NCI-WG) guidelines (2008).9

Table 25. Efficacy Results for Patients with Previously Treated CLL With 17p Deletion by IRC (Study 1)

  Venclexta®
n (%)
N=106 
ORR, n (%) 85 (80.2)
(95% CI) (71.3, 87.3)
CR + CRi, n (%) 8 (7.5)
CR, n (%) 6 (5.7)
CRi, n (%) 2 (1.9)
nPR, n (%) 3 (2.8)
PR, n (%) 74 (69.8)
CI = confidence interval; CR = complete remission; CRi = complete remission with incomplete marrow recovery; IRC = Independent review committee; nPR = nodular partial remission; ORR = overall response rate (CR + CRi + nPR + PR); PR = partial remission.

 

R/R CLL patients with 17p deletion were evaluated for the efficacy of VENCLEXTA® (venetoclax) by overall response rate (ORR). 80.2% ORR was observed. Refer to the drug label for more information.

References

  1. Rai KR, Sawitsky A, Cronkite EP, et al. Clinical staging of chronic lymphocytic leukemia. Blood. 1975;46(2):219-34. 
  2. Binet JL, Auguier A, Dighiero G, et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer. 1981;48(1):198-206.
  3. Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343;1910-6.
  4. Krober A, Seiler T, Benner A, et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood. 2002;100(4):1410-6.
  5. Oscier DG, Gardiner AC, Mould SJ, et al. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood. 2002; 100(4):1177-84.
  6. Shanafelt TD, Witzig TE, Fink SR, et al. Prospective evaluation of clonal evolution during long-term follow-up of patients with untreated early-stage chronic lymphocytic leukemia. J Clin Oncol. 2006; 24(28): 4634-41.
  7. NCCN Clinical Practice Guidelines in Oncology™ NON-HODGKIN’S LYMPHOMAS (Version 2.2016). National Comprehensive Cancer Network, Inc. Available at: NCCN.org.
  8. Tam CS, Shanafelt TD, Wierda WG, et al. De novo deletion 17p13.1 chronic lymphocytic leukemia shows significant clinical heterogeneity: the M. D. Anderson and Mayo Clinic experience. Blood. 2009;114:957-64.
  9. Hallek, M., et al. (2008). “Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updated the National Cancer Institute-Working Group 1996 guidelines.” Blood 111(12): 5446-5456.

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