(448 days)
Not Found
Not Found
No
The document describes a FISH probe kit, which is a laboratory reagent used for genetic testing. The analysis and interpretation of the results are explicitly stated to be performed by a qualified pathologist or cytogeneticist. There is no mention of any automated image analysis, pattern recognition, or data interpretation that would typically involve AI/ML.
No
The device is an in vitro diagnostic (IVD) intended to detect chromosomal abnormalities in bone marrow specimens for the characterization of AML and MDS. It is not used to treat or alleviate a disease condition.
No
The device description explicitly states: "The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic." It is intended for "characterization of patient specimens" and to be used "in conjunction with other clinicopathological criteria."
No
The device is a FISH Probe Kit, which is a physical reagent used in a laboratory process involving fixed bone marrow specimens and fluorescence microscopy. It is not solely software.
Based on the provided information, yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Explicit Statement: The document explicitly states "For in vitro diagnostic use" at the end of the "Intended Use / Indications for Use" section.
- Intended Use: The intended use clearly describes the device as a "fluorescence in situ hybridization (FISH) Test used to detect rearrangement or deletion... in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS)." This aligns with the definition of an IVD, which is used to examine specimens derived from the human body to provide information for diagnostic purposes.
- Specimen Type: The device is used on "fixed bone marrow specimens," which are human biological samples.
- Purpose: The test is indicated for "characterization of patient specimens consistent with World Health Organization (WHO) guidelines... and in conjunction with other clinicopathological criteria." This characterization provides information relevant to the diagnosis and classification of AML and MDS.
- Interpretation by Qualified Personnel: The results are intended to be interpreted by a "qualified pathologist or cytogeneticist," which is typical for IVD tests used in a clinical setting.
While the device is not intended as a stand-alone diagnostic, disease screening, or companion diagnostic, its use in characterizing patient specimens for diagnostic purposes, in conjunction with other clinical information, firmly places it within the realm of In Vitro Diagnostics.
N/A
Intended Use / Indications for Use
The MLL (KMT2A) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the MLL (KMT2A) region on chromosome 11 at location 11g23.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The P53 (TP53) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletion of the P53 (TP53) region on chromosome 7 at location 17p13 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The Del(20q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletion within the long arm of chromosome 20 at locations 20g12 and 20q13.1, in fixed bone marrow specimens from patients with myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The CBFB (CBFB)/MYH11 Translocation, Dual Fusion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the chromosome 16 causing the CBFB-MYH11 (CBFB-MYH11) fusion in fixed bone marrow specimens from patients with acute myeloid leukemia (AML). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The Del(5q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletions within the long arm of chromosome 5 at location 5g31.2 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The Del(7q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletions within the long arm of chromosome 7 at locations 7g22 and 7q31.2 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement involving the AML1 (RUNX1) region on chromosome 21 at location 21g22.1 and the ETO (RUNXITI) region on chromosome 8 at location 8q21.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The EVI1 (MECOM) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement involving the EVII (MECOM) region on chromosome 3 at location 3g26.2, in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
Product codes (comma separated list FDA assigned to the subject device)
QDI
Device Description
Each Cytocell FISH Probe Kit device consists of one vial containing specific probes as described below. The probes are provided premixed in hybridization solution (formamide: dextran sulfate; saline-sodium citrate (SSC)) and are ready to use. The Kit also includes one vial of 4,6-diamidino-2-phenylindole (DAPI) counterstain. The kits are available in a 10-test format.
The MLL (KMT2A) Breakapart FISH Probe Kit consists of an 87kb probe, labeled in Texas red, covering a region telemetric to the MLL (KMT2A) gene including the marker SHGC-111513 and a FITC green probe covering a 170kb region centromeric to the MLL (KMT2A) gene spanning the CD3G and UBE4A genes.
The P53 (TP53) Deletion FISH Probe Kit consists of a 161kb probe, labeled in Texas red, covering the whole P53 (TP53) gene, extending 74kb telomeric to the gene and covering a region centromeric to the gene, to just beyond the marker D17S655; and a probe, labelled in FITC green, covering the chromosome 17 centromere (D17Z1) region.
The Del(20q) Deletion FISH Probe Kit consists of a 331kb probe, labeled in Texas red, covering a region within the PTPRT gene and including the D20S108 marker; and two (141kb and 174kb) probes labeled in FITC green covering the MYBL2 gene and including the D20S150 marker.
The CBFB (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit consists of a 617kb probe, labeled in Texas red, covering a region, within 16q22 including the CBFB gene; and a 621kb probe, labeled in FITC green, covering a region within 16p13.1 including the MYH11 gene.
The Del(5q) Deletion FISH Probe Kit consists of a 186kb probe, labeled in Texas red, covering a region within 5q31.2, including the D5S500 marker; and a 376kb probe, labeled in FITC green, within 5p15.3, including the D5S630 marker.
The Del(7q) Deletion FISH Probe Kit consists of a 396kb probe, labeled in Texas red, covering a region within 7q22 including the telomeric end of the RELN gene and extending beyond the D7S658 marker; and a 203kb probe, labeled in FITC green, covering a region within 7q31.2 including the TES gene.
The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit consists of a 156kb probe labeled in Texas red, centromeric to the AMLI (RUNXI) gene, including the CLIC6 gene; a 169kb probe labelled in Texas red, telomeric to AMLI (RUNXI) gene, extending beyond the marker D21S1921; and two (151kb and 194kb) probes, labeled inn FITC green, on either side of the ETO (RUNXIT) gene.
The EVI1 (MECOM) Breakapart FISH Probe Kit consists of a 158kb probe, labeled in Texas red, telomeric to the D3S4415 marker and including the LRRC34 gene, a FITC green probe covering a 181kb region. including the entire EVI1 (MECOM) gene and flanking regions and a PF-415 blue probe, which covers a 563kb region centromeric to the EVI I (MECOM) gene, including the D3S1614 marker.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Fluorescent in situ hybridization (FISH)
Anatomical Site
Fixed bone marrow specimens
Indicated Patient Age Range
Not Found
Intended User / Care Setting
qualified pathologist or cytogeneticist / 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
Description of the test set: The analytical sensitivity of the probes was established by analyzing interphase nuclei from 25 karyotypically normal bone marrow samples. For two of the nine probes included in this study, AML1 (RUNX1) Breakapart FISH Probe Kit and EVI1 (MECOM) Breakapart FISH Probe Kit, the results generated by the analytical sensitivity study were utilized (25 karyotypically normal bone marrow samples).
Sample size: 25 karyotypically normal bone marrow samples. Each sample was analyzed by 2 independent analysts. Each analyst analyzed 100 nuclei per sample, for a total of 200 nuclei per sample, resulting in 5000 scorable nuclei per probe evaluated.
Data source: Bone marrow samples.
Annotation protocol: Not Found.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Study Type: Analytical performance (Precision/Reproducibility, Detection Limit, Analytical specificity, Assay Cut-off), Clinical Supportive Data (Incidence Rate, Concordance).
Sample Size:
Precision/Reproducibility: ""bone marrow specimens to represent the range of results [two normal (negative), two near cut-off or low positive, and two high positive)]. Specimens were analyzed according to protocol and tested over 5 non-consecutive days in duplicates at " sites to assess precision (n =30 per specimen).
Analytical sensitivity: 25 karyotypically normal bone marrow samples.
Analytical specificity: Five normal male peripheral blood samples ("" metaphase spreads in each sample, of " metaphase spreads if the probe is located on a sex chromosome giving unique loci evaluated for each analysis).
Incidence Rate: Laboratory 1 (GOP) analyzed 100 known and suspected AML and MDS specimens in total. Laboratory 2 (YAL) re-analyzed specimen data (266-742 AML or MDS specimens depending on the probe)
Concordance: Additional clinical data sets (sample size not explicitly stated but reference to "100% agreement" implies a set was used)
AUC: Not found
MRMC: Not found
Stand-alone performance: Not intended for use as a stand-alone diagnostic.
Key Results:
Precision/Reproducibility: Tables 2 through 10 demonstrate that all probe kits had acceptable precision. For two probe kits, additional specimens were re-assessed to confirm acceptable precision at the low end of the enumeration range. Tables 11-19 showed acceptable between-lot reproducibility.
Stability Studies: Real-time stability (24-month shelf-life), Transport stability (no change in performance under stress), Freeze/Thaw stability (10 cycles acceptable), Post-hybridization stability (stable for one month), Photostability (stable up to "" hours).
Analytical sensitivity: All nine probes had an analytical sensitivity greater than 95%, meeting acceptance criteria (Table 21).
Analytical specificity: All probes met acceptance criteria with 100% specificity (Table 22).
Assay Cut-off: Determined using a large patient dataset for seven probes and analytical sensitivity data for two probes.
Incidence Rate: Prevalence rates from GOP and YAL data sets were generally within the expected range from literature (Tables 24-32).
Concordance: 100% agreement between FISH and G-band for AML1/ETO and CBFB/MYH11 probes except for one specimen near the cut-off (Tables 33 & 34).
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Analytical Sensitivity: Greater than 95% for all nine probes (Table 21).
Analytical Specificity: 100% for all components of the probes (Table 22).
Agreement (Reproducibility): Ranges from 27% to 100% for various specimens across different probes, as shown in Tables 2-10 for precision and Tables 11-19 for lot-to-lot reproducibility.
Incidence Rate (Prevalence): Varies by probe and data source, generally consistent with literature ranges (Tables 24-32).
Concordance: 100% agreement between FISH and G-band observed for AML1/ETO and CBFB/MYH11 probes, with one exception noted as being near cut-off.
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.
Not Found
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
§ 864.1880 Fluorescence in situ hybridization (FISH)-based detection of chromosomal abnormalities from patients with hematologic malignancies.
(a)
Identification. A fluorescence in situ hybridization (FISH)-based detection of chromosomal abnormalities from patients with hematologic malignancies is used to detect chromosomal abnormalities in human specimens from patients with hematologic malignancies. The test is indicated for the clinical management of patients consistent with internationally accepted guidelines (e.g., World Health Organization guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues) and in conjunction with other clinical and clinicopathological criteria. The results are to be interpreted by a pathologist or equivalent professional.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Design verification and validation must include:
(i) A detailed description of all probes included in the kit;
(ii) Purpose of each probe;
(iii) Probe molecular specificity;
(iv) Probe specificity;
(v) Probe limits;
(vi) Probe sensitivity;
(vii) Specification of required ancillary reagents, instrumentation, and equipment;
(viii) Specification of the specimen collection, processing, storage and slide preparation methods;
(ix) Specification of the assay procedure;
(x) Specification of control elements that are incorporated into the recommended testing procedures;
(xi) Specification of the criteria for test result interpretation and reporting;
(xii) Documentation demonstrating analytical validation that includes:
(A) Device analytical sensitivity data with a minimum of 25 specimens from karyotypically normal males.
(B) Device analytical specificity data with a minimum of five specimens from karyotypically normal males.
(C) Description of how the clinical threshold was assigned and verification of the assigned clinical threshold.
(D) Device precision/reproducibility data with a minimum of six clinical specimens including two negative specimens, two positive specimens near the clinical decision threshold (cut-off) and two positive specimens. The data must include results obtained from three sites (as applicable), with two operators at each site, with the assay run for a minimum of 3-5 non-consecutive days and each specimen run in duplicate for a minimum of 30 replicates.
(E) Between-reagent lot reproducibility using three reagent lots and three clinical specimens representing negative, near cut-off/low positive, and positive.
(F) Device stability data to include:
(
1 ) Real-time stability,(
2 ) Freeze-thaw stability,(
3 ) Transport and temperature stability, as applicable,(
4 ) Post-hybridization signal stability, and(
5 ) Photostability of probe.(xiii) Documentation demonstrating the clinical validity of the device that includes:
(A) A summary of the prevalence and clinical thresholds reported in three peer-reviewed published literature references for the intended use population of the device and device performance data demonstrating conformance with the published prevalence as reported in peer-reviewed published literature references based on testing clinical specimens, selected without bias (
e.g., consecutively selected) from the intended use population using the specific device seeking marketing clearance. A minimum number of clinical specimens must be tested to ensure sufficient positives are evaluated by the device, or alternatively, in the absence of a sufficient number of positives, an additional comparison of results obtained with the device to clinical truth (e.g., confirmed clinical diagnosis and/or G-banded karyotyping) with an independent specimen set must be conducted.(B) Documentation for peer-reviewed published literature references must include the following elements:
(
1 ) Whether the specific device was used in the literature reference;(
2 ) Number and type of specimens;(
3 ) Target population studied;(
4 ) Upper reference limit; and(
5 ) Prevalence range estimated based on the number of positive probe results.(C) In the absence of clinical data obtained from paragraphs (b)(1)(xiii)(A) and (B) of this section, clinical data obtained from a method comparison to the predicate with positives and negative clinical specimens.
(2) The intended use required on the label under § 809.10(a)(4) of this chapter and on the labeling required under § 809.10(b)(5)(ii) of this chapter must include a statement that “The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.”
(3) The labeling required under § 809.10(b) of this chapter must include information that demonstrates the performance characteristics of the test, including a detailed summary of the performance studies conducted and their results, as described in paragraphs (b)(1)(iv) through (xiii) of this section. The labeling required under § 809.10(b) of this chapter must include the pre-specified acceptance criteria for these performance studies, justification for the pre-specified acceptance criteria, and whether the pre-specified acceptance criteria were met.
(4) The labeling required under § 809.10(b) of this chapter must include the following limiting statements:
(i) “Reporting and interpretation of FISH results should be consistent with professional standards of practice and should take into consideration other clinical and diagnostic information. This kit is intended as an adjunct to other diagnostic laboratory tests and therapeutic action should not be initiated on the basis of the FISH result alone. Failure to adhere to the protocol may affect the performance and lead to false results.”
(ii) “Each lab is responsible for establishing their own cut-off values. Each laboratory should test sufficiently large number of samples to establish normal population distribution of the signal levels and to assign a cut-off value. The product is for professional use only and is intended to be interpreted by a qualified pathologist or cytogeneticist.”
0
EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Cytocell FISH Probe Kits for AML and MDS DECISION SUMMARY
A. DEN Number:
B. Purpose for Submission:
De novo request for evaluation of automatic class III designation for the following Cytocell FISH Probe Kits for AML (Acute Myeloid Leukemia) and/or MDS (Myelodysplastic Syndromes) (refer to the list of devices in Section C below).
C. Measurands:
Chromosomal rearrangements as listed below:
| Device | Chromosomal Rearrangement |
|---|---|
| MLL (KMT2A) Breakapart FISH | |
| Probe Kit | Rearrangement of MLL (KMT2A) region on |
| chromosome 11 at location 11q23.3 | |
| P53 (TP53) Deletion FISH Probe Kit | Deletions of the P53 (TP53) region on chromosome |
| 17, at location 17p13 | |
| Del(20q) Deletion FISH Probe Kit | Deletions within the long arm of chromosome 20 at |
| locations 20q12 and 20q13.1 | |
| CBFβ (CBFB) /MYH11 | |
| Translocation, Dual Fusion FISH | |
| Probe Kit | Rearrangements of chromosome 16 causing the |
| CBFβ-MYH11 (CBFB-MYH11) fusion | |
| Del(5q) Deletion FISH Probe Kit | Deletions within the long arm of chromosome 5 at |
| location 5q31.2 | |
| Del(7q) Deletion FISH Probe Kit | Deletions within the long arm of chromosome 7 at |
| locations 7q22 and 7q31.2 | |
| AML1/ETO (RUNX1/RUNX1T1)) | |
| Translocation, Dual Fusion FISH | |
| Probe Kit | Rearrangements involving the AML1 (RUNX1) |
| region on chromosome 21 at location 21q22.1 and the | |
| ETO (RUNX1T1) region on chromosome 8 at location | |
| 8q21.3 | |
| EVI1 (MECOM) Breakapart FISH | |
| Probe Kit | Rearrangements involving the EVI1 (MECOM) region |
| on chromosome 3 at location 3q26.2 |
1
D. Type of Test:
Fluorescent in situ hybridization (FISH).
E. Applicant:
Cytocell, Ltd
F. Proprietary and Established Names:
- MLL (KMT2A) Breakapart FISH Probe Kit
- P53 (TP53) Deletion FISH Probe Kit
- Del(20q) Deletion FISH Probe Kit
- CBFß (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit
- Del(5q) Deletion FISH Probe Kit
- Del(7q) Deletion FISH Probe Kit
- AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit ●
- EVI1 (MECOM) Breakapart FISH Probe Kit
G. Regulatory Information:
-
- Regulation section:
21 CFR 864.1880
- Regulation section:
-
- Classification:
Class II
- Classification:
-
- Product code(s):
QDI
- Product code(s):
-
- Panel:
Pathology
- Panel:
H. Indications for use:
-
- Indications for use:
The MLL (KMT2A) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the MLL (KMT2A) region on chromosome 11 at location 11g23.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health
- Indications for use:
2
Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The P53 (TP53) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletion of the P53 (TP53) region on chromosome 7 at location 17p13 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The Del(20q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletion within the long arm of chromosome 20 at locations 20g12 and 20q13.1, in fixed bone marrow specimens from patients with myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The CBFB (CBFB)/MYH11 Translocation, Dual Fusion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the chromosome 16 causing the CBFB-MYH11 (CBFB-MYH11) fusion in fixed bone marrow specimens from patients with acute myeloid leukemia (AML). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The Del(5q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletions within the long arm of chromosome 5 at location 5g31.2 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not
3
intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The Del(7q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletions within the long arm of chromosome 7 at locations 7g22 and 7q31.2 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement involving the AML1 (RUNX1) region on chromosome 21 at location 21g22.1 and the ETO (RUNXITI) region on chromosome 8 at location 8q21.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
The EVI1 (MECOM) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement involving the EVII (MECOM) region on chromosome 3 at location 3g26.2, in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
-
- Special conditions for use statement(s) For prescription use. For in vitro diagnostic use
I. Device Description:
Each Cytocell FISH Probe Kit device consists of one vial containing specific probes as described below. The probes are provided premixed in hybridization solution (formamide: dextran sulfate; saline-sodium citrate (SSC)) and are ready to use. The Kit also includes one
4
vial of 4,6-diamidino-2-phenylindole (DAPI) counterstain. The kits are available in a 10-test format.
The MLL (KMT2A) Breakapart FISH Probe Kit consists of an 87kb probe, labeled in Texas red, covering a region telemetric to the MLL (KMT2A) gene including the marker SHGC-111513 and a FITC green probe covering a 170kb region centromeric to the MLL (KMT2A) gene spanning the CD3G and UBE4A genes.
The P53 (TP53) Deletion FISH Probe Kit consists of a 161kb probe, labeled in Texas red, covering the whole P53 (TP53) gene, extending 74kb telomeric to the gene and covering a region centromeric to the gene, to just beyond the marker D17S655; and a probe, labelled in FITC green, covering the chromosome 17 centromere (D17Z1) region.
The Del(20q) Deletion FISH Probe Kit consists of a 331kb probe, labeled in Texas red, covering a region within the PTPRT gene and including the D20S108 marker; and two (141kb and 174kb) probes labeled in FITC green covering the MYBL2 gene and including the D20S150 marker.
The CBFB (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit consists of a 617kb probe, labeled in Texas red, covering a region, within 16q22 including the CBFB gene; and a 621kb probe, labeled in FITC green, covering a region within 16p13.1 including the MYH11 gene.
The Del(5q) Deletion FISH Probe Kit consists of a 186kb probe, labeled in Texas red, covering a region within 5q31.2, including the D5S500 marker; and a 376kb probe, labeled in FITC green, within 5p15.3, including the D5S630 marker.
The Del(7q) Deletion FISH Probe Kit consists of a 396kb probe, labeled in Texas red, covering a region within 7q22 including the telomeric end of the RELN gene and extending beyond the D7S658 marker; and a 203kb probe, labeled in FITC green, covering a region within 7q31.2 including the TES gene.
The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit consists of a 156kb probe labeled in Texas red, centromeric to the AMLI (RUNXI) gene, including the CLIC6 gene; a 169kb probe labelled in Texas red, telomeric to AMLI (RUNXI) gene, extending beyond the marker D21S1921; and two (151kb and 194kb) probes, labeled inn FITC green, on either side of the ETO (RUNXIT) gene.
The EVI1 (MECOM) Breakapart FISH Probe Kit consists of a 158kb probe, labeled in Texas red, telomeric to the D3S4415 marker and including the LRRC34 gene, a FITC green probe covering a 181kb region. including the entire EVI1 (MECOM) gene and flanking regions and a PF-415 blue probe, which covers a 563kb region centromeric to the EVI I (MECOM) gene, including the D3S1614 marker.
K. Standard/Guidance Document Referenced:
Guidance for Industry and FDA Staff - Content and Format for Abbreviated 510(k)s for Early Growth Response 1 (EGR1) Gene Fluorescence In-Situ Hybridization (FISH) Test
5
System for Specimen Characterization Devices
L. Test Principle:
Fluorescence in situ hybridization (FISH) is a technique that allows chromosomal aberrations to be detected on metaphase chromosomes or in interphase nuclei from fixed cytogenetic samples.
Bone marrow cells from patients are attached to microscope slides using standard cytogenetic procedures. After fixation and denaturation of cellular DNA to single-stranded form, target DNA is available for annealing to a similarly denatured, fluorescently labelled DNA probe, which has a complementary sequence. Following hybridization, unbound and nonspecifically bound DNA probe is removed by a series of washes, and the DNA is counterstained for visualization with DAPI, a DNA-specific stain that fluoresces blue. Fluorescence microscopy equipped with appropriate excitation and emission filters then allows the visualization of the hybridized probe on the target material. Two analysts should analyze each sample. Each analyst scores independently 100 nuclei for each sample. In some cases, depending on the number of abnormal nuclei each analyst has seen, a third reader may be required.
The expected signal pattern is dependent on the number of copies of each probe. Enumeration of the signals provide a mechanism for determining absolute copy number of the probe targets and the presence of chromosomal aberrations of interest. Refer to Table 1 -Supported Signal Patterns & Cut-Offs for the expected normal signal pattern and expected abnormal signal pattern for each probe kit (R = red, G = Green, B = blue).
| Catalogue | Name | Negative
Signal | Positive Signal |
|------------|------------------------------------------------------------------------------------------------|--------------------|-----------------|
| USA-LPH013 | MLL (KMT2A) Breakapart | (b)(4) | |
| USA-LPH017 | P53 (TP53) Deletion | | |
| USA-LPH020 | Del (20q) Deletion | | |
| USA-LPH022 | CBFβ (CBFB) /MYH11
Translocation, Dual Fusion | | |
| USA-LPH024 | Del (5q) Deletion | | |
| USA-LPH025 | Del (7q) Deletion | | |
| USA-LPH026 | AML1/ETO
(RUNX1/RUNX1T1)
Translocation, Dual Fusion | | |
| USA-LPH036 | EVI1 (MECOM) Breakapart Inversion
Signal
EVI1 (MECOM) Breakapart
Translocation Signal | | |
Table 1 - Supported Signal Patterns & Cut-Offs
6
M. Performance Characteristics:
Analytical validation was provided in support of each probe kit:
1. Analytical performance:
a. Precision/Reproducibility
Repeatability and reproducibility of each probe kit was assessed using ""bone marrow specimens to represent the range of results [two normal (negative), two near cut-off or low positive, and two high positive]. Specimens were analyzed according to protocol and tested over 5 non-consecutive days in duplicates at " sites to assess precision (n =30 per specimen).
In some cases, to ensure there was ample volume to allow for consistent testing amongst all sites one or more normal samples were combined (pooled) and distributed to testing sites. The majority of the near cut-off and low positive samples were contrived by spiking known normal samples with known positive specimens. All samples were confirmed to be normal by an established alternative cytogenetic method (competitor probe or G-banding) prior to distribution.
Data was analyzed for intra-day, between-day, between-site and total SD.
Two probe kits [CBFB (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit (LPH 022) and EVI1 (MECOM) Breakapart FISH Probe Kit (LPH 036))) had specimens too close the cut-off to assess precision and therefore additional specimens were re-assessed at "" and "" | the cut-off at one site with 2 operators and 5 days in duplicate to confirm acceptable precision at the low end of the enumeration range (n (6)(4) ). The data with the clinical thresholds used in the study are shown in Tables 2 through 10 demonstrate that all probe kits had acceptable precision.
Table 2. Precision of the MLL (KMT2A) Breakapart (cut-off: 3.8%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Between-
site SD | Total
SD |
|----------------|-----------------------------------|-------|-------------------------|------------------|---------------------|---------------------|---------------------|-------------|
| Negative 1 | (b)(4) | | | 100 | 0.00 | 0.00 | 0.00 | 0.00 |
| Negative 1 | | | | 100 | 0.00 | 0.00 | 0.00 | 0.09 |
| Near cut-off 1 | | | | 63 | 0.00 | 0.00 | 1.17 | 1.98 |
| Near cut-off 2 | | | | 90 | 0.00 | 1.69 | 0.87 | 3.12 |
| Positive 1 | | | | 100 | 0.00 | 0.77 | 4.57 | 6.60 |
| Positive 2 | | | | 100 | 0.81 | 0.35 | 3.06 | 4.14 |
N/A = not applicable
7
| Specimen | Mean of abnormal percentage | Range | Agreement replicates | Agreement (%) | Intra-Day SD | Inter-Day SD | Between-site SD | Total SD |
|---|---|---|---|---|---|---|---|---|
| Negative 1 | (b)(4) | 97 | 0.00 | 0.00 | 1.11 | 2.09 | ||
| Negative 1 | 93 | 0.00 | 0.00 | 1.65 | 2.50 | |||
| Near cut-off 1 | 83 | 0.00 | 0.00 | 1.71 | 3.62 | |||
| Near cut-off 2 | 100 | 0.00 | 2.18 | 1.34 | 4.46 | |||
| Positive 1 | 100 | 0.00 | 0.00 | 0.00 | 5.84 | |||
| Positive 2 | 100 | 0.00 | 0.92 | 4.18 | 6.04 |
Table 3. Precision of the P53 (TP53) Deletion (cut-off: 6.8%)
Table 4. Precision of the Del (20q) Deletion (cut-off: 5.7%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Between-
site SD | Total
SD |
|----------------|-----------------------------------|-------|-------------------------|------------------|---------------------|---------------------|---------------------|-------------|
| Negative 1 | (b)(4) | | | 100 | 0.00 | 0.48 | 0.00 | 1.41 |
| Negative 1 | | | | 100 | 0.00 | 0.33 | 0.67 | 1.01 |
| Near cut-off 1 | | | | 80 | 0.00 | 0.00 | 0.00 | 4.45 |
| Near cut-off 2 | | | | 100 | 0.00 | 2.01 | 2.79 | 5.31 |
| Positive 1 | | | | 100 | 0.00 | 0.00 | 0.00 | 6.93 |
| Positive 2 | | | | 100 | 0.00 | 0.00 | 0.48 | 4.96 |
Table 5. Precision of the CBFβ (CBFB) /MYH11 Translocation, Dual Fusion (cut-off: 2.3%)
| Specimen | Mean of abnormal percentage | Range | Agreement replicates | Agreement (%) | Intra-Day SD | Inter-Day SD | Between-site SD | Total SD |
|---|---|---|---|---|---|---|---|---|
| Negative 1 | (b)(4) | 100 | 0.00 | 0.00 | 0.00 | 0.00 | ||
| Negative 1 | 100 | 0.00 | 0.00 | 0.00 | 0.09 | |||
| Near cut-off 1 | 27 | 0.00 | 0.51 | 0.00 | 1.30 | |||
| Near cut-off 2 | 43 | 0.75 | 0.00 | 0.10 | 2.47 | |||
| Positive 1 | 100 | 0.00 | 0.84 | 0.64 | 4.55 | |||
| Positive 2 | 100 | 0.00 | 1.76 | 3.20 | 7.50 |
N/A = not applicable
8
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Total
SD |
| ---------- | ----------------------------------- | ------- | ------------------------- | ------------------ | --------------------- | --------------------- | ------------- |
|---|
100
100
0.56
0.00
0.25
0.00
1.34
2.42
| Table 5.a. Additional supporting date for precision of the CBFβ (CBFB) /MYH11 | |
|---|---|
| Translocation, Dual Fusion |
| Table 6. Precision of the Del (5q) Deletion (cut-off: 6.3%) | ||
|---|---|---|
(b)(4)
Near cut-off 1 Near cut-off 2
| Specimen | Mean of
abnormal | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Between-
site SD | Total
SD |
|----------------|---------------------|-------|-------------------------|------------------|---------------------|---------------------|---------------------|-------------|
| | (b)(4) | | | | | | | |
| Negative 1 | | | | 100 | 0.32 | 0.58 | 0.73 | 1.25 |
| Negative 1 | | | | 100 | 0.00 | 0.06 | 1.30 | 1.59 |
| Near cut-off 1 | | | | 80 | 0.00 | 2.47 | 3.67 | 5.85 |
| Near cut-off 2 | | | | 97 | 2.06 | 0.00 | 1.08 | 4.49 |
| Positive 1 | | | | 100 | 1.93 | 3.27 | 0.89 | 7.04 |
| Positive 2 | | | | 100 | 0.00 | 1.21 | 3.18 | 5.09 |
Table 7. Precision of the Del (7q) Deletion (cut-off: 7.4%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Between-
site SD | Total
SD |
|----------------|-----------------------------------|-------|-------------------------|------------------|---------------------|---------------------|---------------------|-------------|
| Negative 1 | (b)(4) | | | 100 | 0.00 | 0.11 | 0.87 | 1.12 |
| Negative 1 | | | | 100 | 0.00 | 0.00 | 0.98 | 1.67 |
| Near cut-off 1 | | | | 100 | 0.00 | 0.30 | 2.54 | 4.86 |
| Near cut-off 2 | | | | 100 | 0.39 | 0.00 | 2.28 | 4.21 |
| Positive 1 | | | | 100 | 0.00 | 0.00 | 3.00 | 5.09 |
| Positive 2 | | | | 100 | 0.00 | 0.31 | 2.16 | 3.79 |
Table 8. Precision of the AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion (cutoff: 2.3%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Between-
site SD | Total
SD |
|----------------|-----------------------------------|-------|-------------------------|------------------|---------------------|---------------------|---------------------|-------------|
| Negative 1 | (b)(4) | | | 100 | 0.00 | 0.00 | 0.00 | 0.00 |
| Negative 1 | | | | 100 | 0.00 | 0.00 | 0.00 | 0.00 |
| Near cut-off 1 | | | | 80 | 0.23 | 0.00 | 0.12 | 1.72 |
| Near cut-off 2 | | | | 97 | 1.57 | 0.00 | 0.82 | 2.40 |
| Positive 1 | | | | 100 | 0.00 | 1.06 | 0.00 | 8.30 |
| Positive 2 | | | | 100 | 0.25 | 0.00 | 0.00 | 6.25 |
N/A = not applicable
9
Table 9. Precision of the EVI1 (MECOM) Breakapart_Inversion (cut-off: 4.0%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Between-
site SD | Total
SD |
|----------------|-----------------------------------|-------|-------------------------|------------------|---------------------|---------------------|---------------------|-------------|
| Negative 1 | (b)(4) | | | 100 | 0.24 | 0.40 | 0.54 | 1.02 |
| Negative 1 | | | | 100 | 0.52 | 0.00 | 0.26 | 1.24 |
| Near cut-off 1 | | | | 53 | 0.00 | 1.20 | 0.00 | 3.49 |
| Near cut-off 2 | | | | 73 | 0.00 | 1.25 | 1.06 | 4.13 |
| Positive 1 | | | | 100 | 0.68 | 3.01 | 7.05 | 11.93 |
| Positive 2 | | | | 100 | 2.42 | 0.00 | 5.56 | 13.26 |
- these two specimens are too close to cut-of to allow assessment of precision An additional onesite precision study with low positive specimens is provided below.
Table 9.a. Additional supporting date for precision of the EVI1 (MECOM) Breakapart Inversion
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
Day
SD | Inter-
Day
SD | Total
SD |
|----------------|-----------------------------------|-------|-------------------------|------------------|---------------------|---------------------|-------------|
| Near cut-off 1 | (b)(4) | | | 100 | 0.26 | 1.78 | 2.75 |
| Near cut-off 2 | | | | 100 | 0.00 | 0.00 | 2.11 |
Table 10. Precision of the EVI1 (MECOM) Breakapart Translocation (cut-off: 4.0%)
| Specimen | Mean of | Range | Agreement | Agreement | Intra- | Inter- | Between- | Total |
|---|---|---|---|---|---|---|---|---|
| abnormal | replicates | %) | Day | Day | site SD | SD | ||
| percentage | SD | SD | ||||||
| Negative 1 | (b)(4) | 100 | 0.00 | 0.00 | 0.95 | 1.02 | ||
| Negative 1 | 100 | 0.00 | 0.32 | 0.84 | 1.09 | |||
| Near cut-off 1 | 97 | 0.00 | 1.47 | 2.87 | 4.27 | |||
| Near cut-off 2 | 100 | 0.00 | 0.00 | 1.27 | 3.38 | |||
| Positive 1 | 100 | 0.00 | 0.00 | 3.34 | 6.79 | |||
| Positive 2 | 100 | 1.11 | 0.37 | 0.00 | 6.78 |
Between- Lot reproducibility:
Three reagent lots were evaluated using one normal, one low positive and one high positive specimens were tested with " replicates per lot to assess lot to lot variation (total of [" replicates evaluated). Intra-lot, inter-lot and total SD for each lot are shown in Tables 11-19 below.
10
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|---------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 100 | 0.0 | 0.00 | 0.00 |
| Near cut-off | | | | 100 | 0.9 | 1.43 | 2.09 |
| Positive | | | | 100 | 0.0 | 1.46 | 3.97 |
| N/A = not app | | | | | | | |
Table 11. Lot to lot reproducibility of the MLL (KMT2A) Breakapart (cut-off: 3.8%)
Table 12. Lot to lot reproducibility of the P53 (TP53) Deletion (cut-off: 6.8%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 100 | 0.00 | 0 | 1.20 |
| Near cut-off | | | | 100 | 0.00 | 0 | 4.85 |
| Positive | | | | 100 | 2.25 | 0 | 2.76 |
Table 13. Lot to lot reproducibility of the Del (20q) Deletion (cut-off: 5.7%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 92 | 0.00 | 0.00 | 2.17 |
| Near cut-off | | | | 67 | 1.41 | 0.00 | 3.25 |
| Positive | | | | 100 | 0.00 | 2.62 | 4.95 |
Table 14. Lot to lot reproducibility of the CBFβ (CBFB) /MYH11 Translocation, Dual Fusion (cut-off: 2.3%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 100 | 0.00 | 0 | 0.00 |
| Near cut-off | | | | 33 | 1.12 | 0 | 2.06 |
| Positive | | | | 100 | 0.00 | 0 | 7.69 |
Table 15. Lot to lot reproducibility of the Del (5q) Deletion (cut-off: 6.3%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 83 | 0.98 | 0.49 | 2.09 |
| Near cut-off | | | | 92 | 0.00 | 0.00 | 4.06 |
| Positive | | | | 100 | 0.00 | 1.40 | 3.66 |
11
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 100 | 0.00 | 0.08 | 0.94 |
| Near cut-off | | | | 100 | 0.00 | 0.00 | 5.78 |
| Positive | | | | 100 | 1.82 | 0.00 | 4.03 |
Table 16. Lot to lot reproducibility of the Del (7q) Deletion (cut-off: 7.4%)
Table 17. Lot to lot reproducibility of the AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion (cut-off: 2.3%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 100 | 0.00 | 0.00 | 0.14 |
| Near cut-off | | | | 100 | 0.87 | 0.58 | 1.84 |
| Positive | | | | 100 | 0.80 | 1.18 | 4.85 |
Table 18. Lot to lot reproducibility of the EVI1 (MECOM) Breakapart Inversion (1RG/1B/1RGB) (cut-off: 4.0%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | ' Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|--------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 92 | 0.00 | 0.00 | 2.52 |
| Near cut-off | | | | 67 | 1.63 | 2.82 | 6.29 |
| Positive | | | | 100 | 1.80 | 3.01 | 6.99 |
Table 19. Lot to lot reproducibility of the EVI1 (MECOM) Breakapart Translocation (1R/1GB/1RGB) (cut-off: 4.0%)
| Specimen | Mean of
abnormal
percentage | Range | Agreement
replicates | Agreement
(%) | Intra-
lot SD | Inter-
lot SD | Total
SD |
|--------------|-----------------------------------|-------|-------------------------|------------------|------------------|------------------|-------------|
| Negative | (b)(4) | | | 100 | 0.25 | 0.00 | 0.70 |
| Near cut-off | | | | 100 | 0.96 | 1.80 | 3.55 |
| Positive | | | | 100 | 4.67 | 3.74 | 7.62 |
b. Linearity/assay Reportable Range:
Not applicable
12
- c. Traceability, Stability, Expected Values (controls, calibrators, or methods):
Several stability studies were conducted in support of the probe kits. The specifics of each study are described below and summarized in Table 20 below.
Real-time Kit stability study:
The real-time stability of the probes was assessed using an isochronal study whereby probe lots of differing ages were assessed at the same time on identical samples. One normal, one low positive, and one high positive specimens were each tested in triplicate on each lot. Three lots of probes with minimum age of 25 months were tested in support of a 24-month shelf-life claim. Probe lots of intermediate ages between 0 and 25 months were also included to provide interim data. The result supported the claimed shelf life of 24 months when stored at -20 C.
Transport Stability
The transport study was conducted by stressing probes under potential extremes of transport conditions. One normal, one low positive, and one high positive specimens were each tested in triplicate using one lot of probe. There was no change in device performance under the stress conditions.
Freeze/Thaw Stability
The freeze/thaw stability was assessed by testing the probes at 0, 5, and 11 freeze/thaw cycles to support the "" cycles claim. One normal, one low positive, and one high positive specimens were each tested in triplicate using one lot of probe. Ten freeze-thaw cycles were found to be acceptable for this kit.
Post Hybridization Stability
The post hybridization stability was assessed by re-analyzing hybridized slides at '0)(4) weeks after hybridization to support the 1month claim. One normal, one low and" positive, and one high positive specimens were each tested in triplicate using one lot of probe. The post-hybridization signal was determined to be stable to one month.
Photostabilitv
The photostability of the probes was assessed by exposing probes to light for 0, 24, and "" hours to support the limited exposure to light claim. One normal, one low positive, and one high positive specimens were each tested in triplicate using one lot of probe. The photo-stability of slides was supported to "" hours. Prolonged exposure of slides to light should be avoided.
Table 20. Summary of stability studies
13
| Test | Samples | Lots | Storage | Intervals | Baseline |
|---|---|---|---|---|---|
| Real Time | |||||
| Stability | (b)(4) | -20 °C | (b)(4) | ||
| Transport | |||||
| Stability | 2 weeks at | ||||
| +40 °C, then | |||||
| -20 °C | |||||
| Freeze/Thaw | |||||
| Stability | -20 °C | ||||
| Post | |||||
| Hybridizatio | |||||
| Stability | Refrigerator | ||||
| temperature | |||||
| (in the dark) | |||||
| Photostabilit | Room | ||||
| Temperature | |||||
| (fluorescent | |||||
| light) |
BM - bone marrowN = negative; LP = low positive; HP = high positive; F/T = freeze thaw; t = time point
d. Detection Limit:
The analytical sensitivity of the probes was established by analyzing interphase nuclei from 25 karyotypically normal bone marrow samples. Each sample was analyzed by 2 independent analysts and the signal pattern of each interphase was recorded. Each analyst analyzed 100 nuclei per sample, for a total of 200 nuclei per sample, resulting in 5000 scorable nuclei per probe evaluated. All nine probes evaluated in this study have an analytical sensitivity of greater than 95%, meeting the required acceptance criteria for analytical sensitivity (Table 21).
| Table 21. Analytical sensitivity of the probes | |||
|---|---|---|---|
| ------------------------------------------------ | -- | -- | -- |
| Probe name | Number of
interphase nuclei
with the expected
normal signal
pattern | Total number
of interphase
nuclei
analyzed | Analytical
sensitivity
(%) | 95%
Confidence
interval (%) |
|---------------------------------|---------------------------------------------------------------------------------|-----------------------------------------------------|----------------------------------|-----------------------------------|
| MLL (KMT2A)
Breakapart probe | (b)(4) | | | |
| P53 (TP53) Deletion
Probe | | | | |
14
| Del(20q) Deletion
| Probe | 4924 | 5000 | 98.48 | 98.1-98.78 |
|---|---|---|---|---|
| CBFB | ||||
| (CBFB)/MYH11 | ||||
| Translocation, Dual | ||||
| Fusion Probe | (b)(4) | |||
| Del(5q) Deletion Probe | ||||
| Del(7q) Deletion Probe | ||||
| AML1/ETO | ||||
| (RUNX1/RUNX1T1) | ||||
| Translocation, Dual | ||||
| Fusion Probe | ||||
| EVI1 (MECOM) | ||||
| Breakapart Probe |
- e. Analytical specificity:
Analytical specificity is defined as the percentage of signals that hybridize to the correct locus and no other location. To establish the probe analytical specificity, each probe was assessed to verify that it hybridizes to the specific target chromosome location. Five(b) normal male peripheral blood samples ("" metaphase spreads in
each sample, of " metaphase spreads if the probe is located on a sex chromosome giving(0)(9) unique loci evaluated for each analysis). The analytical specificity of each product 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. All probes met the required acceptance criteria for analytical specificity with all components having a specificity of 100%. (Table 22).
| Table 22. Analytical specificity of the probes | ||
|---|---|---|
| Probe | Target | Number of
metaphase
chromosomes
hybridized | Number of
correct
hybridized
loci | Analytical
Specificity | 95%
Confidence
Interval |
|---------------------------------------|--------|-----------------------------------------------------|--------------------------------------------|---------------------------|-------------------------------|
| MLL (KMT2A) Breakapart FISH Probe Kit | (b)(4) | | | | |
15
| Probe | Target | Number of
metaphase
chromosomes
hybridized | Number of
correct
hybridized
loci | Analytical
Specificity | 95%
Confidence
Interval |
|----------------------------------------|--------|-----------------------------------------------------|--------------------------------------------|---------------------------|-------------------------------|
| (b)(4) | | | | | |
| EVI1 (MECOM) Breakapart FISH Probe Kit | | | | | |
| EVI1, Red | 3q26.2 | 200 | 200 | 100% | 98.12% - 100% |
| EVI1, Green | 3q26.2 | 200 | 200 | 100% | 98.12% - 100% |
| EVI1, Blue | 3q26.2 | 200 | 200 | 100% | 98.12% - 100% |
Assay Cut-off (Upper Reference Limit): f.
The assay cut off was assigned as follows: For seven of the nine probes, a central clinical laboratory generated the clinical cut-off using a large patient dataset exceeding the ACMG minimum sample size guidelines. In addition, analytical sensitivity data from 25 karyotypically normal bone marrow samples was used to confirm the normal cut-offs. None of the 25 normal samples showed an abnormal signal pattern at or above the normal cut-offs confirming the clinical cut-off established.
For two of the nine probes included in this study, AML1 (RUNX1) Breakapart FISH Probe Kit and EVI1 (MECOM) Breakapart FISH Probe Kit, the results generated by the analytical sensitivity study were utilized (25 karyotypically normal bone marrow samples). In this case: ≥ 25 karyotypically normal bone marrow samples were enumerated and the normal cut-offs were calculated using the ß inverse function (BETAINV) (Table 23).
16
An analytical result above the normal cut-off (upper reference limit) is deemed to be positive. Conversely, an analytical result below the cut-off is deemed to be negative.
| Name | Negative
Signal | Positive Signal | Cut-off |
|-----------------------------------------------------------|--------------------|-----------------|---------|
| MLL (KMT2A) Breakapart | (b)(4) | 1F/1R/1G | 3.8% |
| P53 (TP53) Deletion | | 1R/2G | 6.8% |
| Del (20q) Deletion | | 1R/1G | 5.7% |
| CBFβ (CBFB) /MYH11
Translocation, Dual Fusion | | 2F/1R/1G | 2.3% |
| Del (5q) Deletion | | 1R/2G | 6.3% |
| Del (7q) Deletion | | 1R/1G | 7.4% |
| AML1/ETO
(RUNX1/RUNX1T1)
Translocation, Dual Fusion | | 2F/1R/1G | 2.3% |
| EVI1 (MECOM) Breakapart
Inversion Signal | | 1RG/1B/1RGB | 4.0% |
| EVI1 (MECOM) Breakapart
Translocation Signal | | 1R/1GB/1RGB | 4.0% |
Table 23: Supported Signal Patterns & Cut-Offs
- g. Probe limit
Not applicable. The probes are intended to be used only at the concentration provided and are not intended to be diluted.
Comparison Studies: 2.
-
a. Method comparison with predicated device:
Not applicable -
b. Matrix Comparison
Not applicable -
- Clinical Studies:
- a. Clinical sensitivity Not applicable
- b. Clinical specificity Not applicable
17
C. Other clinical supportive data
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS)
AML and MDS are neoplastic hematological disorders that arise from myeloid progenitor cells in the bone marrow. AML is characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow or other tissues, while MDS is characterized by the simultaneous proliferation and apoptosis of hematopoietic cells.
Refer to the WHO Guidelines for the most up-to-date use of the probes.
Incident rate
Reference to the clinical significance in WHO guidelines and peer-reviewed published papers (3 for AML and 3 for MDS) were provided to support the clinical validity of the device in characterizing bone marrow specimens from patients with AML and/or MDS. Prevalence rates based on clinical thresholds defined in the literature were reviewed and summarized. Clinical specimens were tested in using the Cytocell AML FISH probe sets and assigned cut-offs and the incidence rates were compared to literature. Laboratory 1 (GOP) analyzed 100 known and suspected AML and MDS specimens in total. Laboratory 2 (YAL) re-analyzed specimen data (266-742 AML or MDS specimens depending on the probe) tested in their laboratory based on the assigned cut-off. See Table 24 for the summary of results and Tables 25-33 for individual probe results.
| Probes/
Rearrangement | Expected
Prevalence
Rate from
Literature | GOP data set
Prevalence
(95% CI) | YAL data set
Prevalence (95% CI) |
|---------------------------------------------|---------------------------------------------------|----------------------------------------|-------------------------------------|
| MLL (KMT2A)
Breakapart | (b)(4) | | |
| P53 (TP53) deletion | | | |
| Del(20q) deletion | | | |
| CBFB/MYH11
translocation, dual
fusion | | | |
| Del(5q) deletion | | | |
18
| (b)(4) | |
|---|---|
| Del(7q) deletion | |
| AML/ETO | |
| (RUNX1/RUNX1T1) | |
| dual fusion, | |
| translocation | |
| EVI1 (MECOM) | |
| Breakapart |
N/A = not applicable as data was not obtained from this site for this probe.
| Table 25. MLL (KMT2A) breakapart probe incidence rate | |
|---|---|
| ------------------------------------------------------- | -- |
| Condition | Literature
Source 1
Schanz et
al. | Literature
Source 2
Zhao et al. | Literature
Source 3
Wang et al. | Literature
Source 4
Papaemmanuil
et al. | Literature
Source 5
Grimwade
et al. | Literature
Source 6
Bacher et
al. | Data
Source 1
GOP | Data
Source 2
YAL |
|-----------------------------------------------------------------------------------------------------|--------------------------------------------|---------------------------------------|---------------------------------------|--------------------------------------------------|----------------------------------------------|--------------------------------------------|------------------------------------------------------------------------------|--------------------------------------------------------------------|
| Was the
specific
device under
review in the
submission
used in the
study? | No | No | No | No | No | No | Yes | Yes |
| Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| Target
population
(disease
status) | Confirmed
MDS | Confirmed
MDS | Confirmed
MDS | Confirmed
AML | Confirmed
AML | Confirmed
AML | Known or
suspected
MDS or
AML | Known or
suspected
MDS or
AML |
| Upper
reference
limit - 'Cut-
off value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | N/A | N/A | N/A | (b)(4)
1R1G1F
patterns
per 200
scoreable
interphase
nuclei | 1R1G1F
patterns
per 200
scoreable
interphase
nuclei |
19
| Total Number
of specimens
tested for
each claimed
| type | 2902 | 2404 | 435 | 1540 | 5876 | 2235 | 100 | 413 |
|---|---|---|---|---|---|---|---|---|
| Number of | ||||||||
| specimens | ||||||||
| with a | ||||||||
| positive probe | ||||||||
| result | N/A | N/A | N/A | N/A | N/A | N/A | 2 | 6 |
| Range of | ||||||||
| positive probe | ||||||||
| results | N/A | N/A | N/A | N/A | N/A | N/A | (b)(4) | |
| Source | ||||||||
| incidence rate | ||||||||
| for | ||||||||
| rearrangement | ||||||||
| (95% CI) | 0.2% | 1.24% | 1.9% | 2.86% | 4.5% | 2.72% | ||
| *'11q | ||||||||
| abnormalities' | Expected range from | |||||||
| literature: |
Table 26. P53(TP53) deletion probe incidence rate
| Condition | Literature
Source 1
Schanz et al. | Literature
Source 2
Bernasconi et al. | Literature
Source 3
Wang et al. | Literature
Source 4
Papaemmanuil et al. | Literature
Source 5
Grimwade et al. | Literature
Source 6
Lazarevic et al. | Data
Source 1
GOP |
|-----------------------------------------------------------------------------------------------------|-----------------------------------------|---------------------------------------------|---------------------------------------|-----------------------------------------------|-------------------------------------------|--------------------------------------------|-------------------------|
| Was the
specific
device under
review in the
submission
used in the
study? | No | No | No | No | No | No | Yes |
| Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
20
| Target
population
(disease
status) | Confirmed
MDS | Confirmed
MDS | Confirmed
MDS | Confirmed
AML | Confirmed
AML | Confirmed
AML | Known or
suspected
MDS or
AML |
|-------------------------------------------------------------------------------------------|------------------|------------------|------------------|------------------|------------------|------------------|------------------------------------------------------------------------------------|
| Upper
reference limit
- 'Cut-off
value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | N/A | N/A | N/A | (b)(4)
r
14 1R2G
patterns
per 200
scoreable
interphase
nuclei |
| Total Number
of specimens
tested for each
claimed type | 2902 | 331 | 435 | 1540 | 5876 | 3251 | 100 |
| Number of
specimens
with a positive
probe result | N/A | N/A | N/A | N/A | N/A | N/A | 5 |
| Range of
positive probe
results | N/A | N/A | N/A | N/A | N/A | N/A | (b)(4) |
| Source
incidence rate
for
rearrangement
(95% CI) | 0.6% | 1.8% | 1.9% | 4% | 4% | 8.8% | Expected range from
literature: |
Table 27. Del(20q) deletion probe incidence rate
| Condition | Literature
Source 1
Schanz et
al. | Literature
Source 2
Haas et al. | Literature
Source 3
Wang et
al. | Data Source
1
GOP | Data Source
2
YAL |
|-----------------------------------------------------------------------------------------------------|--------------------------------------------------|----------------------------------------------|-------------------------------------------|------------------------------------------------------------------------------|------------------------------------------------------------------------------|
| Was the
specific
device under
review in the
submission
used in the
study? | No | No | No | Yes | Yes |
| Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes |
| Target
population
(disease
status) | Confirmed
MDS | Confirmed
MDS | Confirmed
MDS | Known or
suspected | Known or
suspected |
| Upper
reference
limit - 'Cut-
off value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | (b)(4) | |
| Total Number
of specimens
tested for
each claimed
type | 2902 | 2072 | 435 | 100 | 742 |
| Number of
specimens
with a
positive probe
result | N/A | N/A | N/A | 5 | 9 |
| Range of
positive probe
results | N/A | N/A | N/A | (b)(4) | |
| Source
incidence rate
for
rearrangement
(95% CI) | 1.7% | 3.6% | 6.6% | | |
| Condition | Literature
Source 1
Papaemmanuil
et al. | Literature
Source 2
Grimwade
et al. | Literature
Source 3
Dores et
al. | Data Source 1
GOP | Data Source 2
YAL |
| Was the
specific device
under review
in the
submission
used in the
study? | No | No | No | Yes | Yes |
| Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes |
| Target
population
(disease status) | Confirmed
AML | Confirmed
AML | Confirmed
AML | Known or
suspected MDS
or AML | Known or
suspected
MDS or AML |
| Upper
reference limit
- 'Cut-off
value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | 2.3% or 5
1R1G2F
patterns per
200 scoreable
interphase
nuclei | 2.3% or 5
1R1G2F
patterns per
200 scoreable
interphase
nuclei |
| Total Number
of specimens
tested for each
claimed type | (b)(4) | | | | |
| Number of
specimens
with a positive
probe result | N/A | N/A | N/A | 2 | 7 |
| Range of
positive probe
results | N/A | N/A | N/A | 83.0%-93.0% | 14%-99.5% |
| Source
incidence rate
for
rearrangement
%
(95% CI) | 5.26% | 5% | 1.04% | 2%
(0.24% to
7.04%) | 2.63%
(1.06% to
5.35%) |
| | | Expected range from
literature: | | 1.04% - 5.26% | |
21
22
Table 28. CBFB/MYH11 translocation, dual fusion probe incidence rate
23
Table 29. Del(5q) deletion probe incidence rate
| Condition | Literature
Source 1
Schanz et
al. | Literature
Source 2
Haase et
al. | Literature
Source 3
Bernasconi
et al. | Literature
Source 4
Papaemmanuil
et al. | Literature
Source 5
Grimwade
et al. | Literature
Source 6
Sanderson
et al. | Data
Source 1
GOP | Data
Source 2
YAL |
|-----------------------------------------------------------------------------------------------------|--------------------------------------------|-------------------------------------------|------------------------------------------------|--------------------------------------------------|----------------------------------------------|-----------------------------------------------|--------------------------------------------------------------------------------|--------------------------------------------------------------------------------|
| Was the
specific
device under
review in the
submission
used in the
study? | No | No | No | No | No | No | Yes | Yes |
| Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| Target
population
(disease
status) | Confirmed
MDS | Confirmed
MDS | Confirmed
MDS | Confirmed
AML | Confirmed
AML | Confirmed
AML | Known or
suspected
MDS or
AML | Known or
suspected
MDS or
AML |
| Upper
reference
limit - 'Cut-
off value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | N/A | N/A | N/A | 6.3% or 13
1R2G
patterns
per 200
scoreable
interphase
nuclei | 6.3% or 13
1R2G
patterns
per 200
scoreable
interphase
nuclei |
| Total Number
of specimens
tested for
each claimed | 2902 | 2072 | 331 | 1540 | 5876 | 1709 | 100 | 723 |
24
| type | ||||||||
|---|---|---|---|---|---|---|---|---|
| Number of | ||||||||
| specimens | ||||||||
| with a | ||||||||
| positive probe | ||||||||
| result | N/A | N/A | N/A | N/A | N/A | N/A | 9 | 73 |
| Range of | ||||||||
| positive probe | ||||||||
| results | N/A | N/A | N/A | N/A | N/A | N/A | 8%-98% | 8%-99.5% |
| Source | ||||||||
| incidence rate | ||||||||
| for | ||||||||
| rearrangement | ||||||||
| (95% CI) | 8% | 15.1% | 16% | 6% | 5% | 11% | 9% | |
| (4.20% to | ||||||||
| 6.40%) | 10.9% | |||||||
| (8% to | ||||||||
| 12.53%) | ||||||||
| Expected range from | ||||||||
| literature: | ||||||||
| 5% - 16% |
Table 30. Del(7q) deletion probe incidence rate
| Condition | Literature
Source 1
Schanz et
al. | Literature
Source 2
Haase et
al. | Literature
Source 3
Bernasconi
et al. | Literature
Source 4
Papaemmanuil
et al. | Literature
Source 5
Grimwade
et al. | Literature
Source 6
Sanderson
et al. | Data Source
1
GOP | Data Source
2
YAL |
|-----------------------------------------------------------------------------------------------------|--------------------------------------------|-------------------------------------------|------------------------------------------------|--------------------------------------------------|----------------------------------------------|-----------------------------------------------|----------------------------------------|----------------------------------------|
| Was the
specific
device under
review in the
submission
used in the
study? | No | No | No | No | No | No | Yes | Yes |
| Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| Target
population
(disease
status) | Confirmed
MDS | Confirmed
MDS | Confirmed
MDS | Confirmed
AML | Confirmed
AML | Confirmed
AML | Known or
suspected
MDS or
AML | Known or
suspected
MDS or
AML |
25
| Upper
reference
limit - 'Cut-
off value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | N/A | N/A | N/A | 7.4% or 15
1R1G
patterns per
200
scoreable
interphase
nuclei | 7.4% or 15
1R1G
patterns per
200
scoreable
interphase
nuclei |
|--------------------------------------------------------------------------------------------|------|-------|------|------|-----------------------------------|------|--------------------------------------------------------------------------------|--------------------------------------------------------------------------------|
| Total Number
of specimens
tested for
each claimed
type | 2902 | 2072 | 331 | 1540 | 5876 | 1709 | 100 | 746 |
| Number of
specimens
with a
positive probe
result | N/A | N/A | N/A | N/A | N/A | N/A | 4 | 48 |
| Range of
positive probe
results | N/A | N/A | N/A | N/A | N/A | N/A | 21.5%-96% | 9%-98% |
| Source
incidence rate
for
rearrangement
(95% CI) | 3.6% | 11.1% | 7.8% | 5.7% | 8% | 9% | 4%
(1.10% to
9.93%) | 6.43%
(4.78% to
8.44%) |
| | | | | | Expected range from
literature | | 3.6% - 11.1% | |
Table 31. AML1/ETO (RUNX1/RUNX1T1) dual fusion translocation probe incidence rate
| Condition | Literature
Source 1
Papaemmanuil
et al. | Literature
Source 2
Grimwade
et al. | Literature
Source 3
Dores et al. | Data Source
1
GOP | Data Source
2
YAL | Condition | Literature
Source 1
Schanz et
al | Literature
Source 2
Haase | Literature
Source 3
Pozdnyakova
et al | Literature
Source 4
Papaemmanuil
et al | Literature
Source 5
Grimwade
et al | Literature
Source 6
Byrd et al | Data Source 1
GOP |
|-----------------------------------------------------------------------------------------------------|--------------------------------------------------|----------------------------------------------|----------------------------------------|---------------------------------------------------------------------------------|------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------|-------------------------------------------|---------------------------------|------------------------------------------------|-------------------------------------------------|---------------------------------------------|--------------------------------------|------------------------------------------------------------------------------------------------------|
| Was the
specific device
under review
in the
submission
used in the
study? | No | No | No | Yes | Yes | Was the
specific
device under
review in the
submission
used in the
study? | No | No | No | No | No | No | Yes |
| Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes | Was the
specimen type
in the study
representative
of the claimed
specimen
type(s) | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| Target
population
(disease status) | Confirmed
AML | Confirmed
AML | Confirmed
AML | Known or
suspected
MDS or
AML | Known or
suspected
MDS or
AML | Target
population
(disease
status) | Confirmed
MDS | Confirmed
MDS | Confirmed
MDS | Confirmed
AML | Confirmed
AML | Confirmed
AML | Known or
suspected
MDS or AML |
| Upper
reference limit
- 'Cut-off
value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | 2.3% or 5
1R1G2F
patterns per
200
scoreable
interphase
nuclei | 2.3% or 5
1R1G2F
patterns per
200 scoreable
interphase
nuclei | Upper
reference
limit - 'Cut-
off value'
(percentage
and per 200
nuclei) | N/A | N/A | N/A | N/A | N/A | N/A | 4% or 8
1RG/1B/1RGB
or
1R/1GB/1RGB
patterns per
200 scoreable
interphase
nuclei |
| Total Number
of specimens
tested for each
claimed type | 1540 | 5876 | 19497 | 100 | 414 | Total Number
of specimens
tested for
each claimed
type | 2902 | 2072 | 1029 | 1540 | 5876 | 1213 | 100 |
| Number of
specimens with
a positive
probe result | N/A | N/A | N/A | 0 | 6 | Number of
specimens
with a
positive probe
result | N/A | N/A | N/A | N/A | N/A | N/A | 4 |
| Range of
positive probe
results | N/A | N/A | N/A | N/A | 69.5%-98.5% | | | | | | | | |
| Source
incidence rate
for
rearrangement
%
(95% CI) | 3.8% | 7% | 1.6% | 0%
(0.00% to
3.62%) | 1.45%
(0.53% to
3.13%) | | | | | | | | |
| | | Expected range from
literature | | | 1.6% - 7.0% | | | | | | | | |
26
27
Table 32. EVI1 Breakapart Probe incidence rate
28
| Range of
positive probe
| results | N/A | N/A | N/A | N/A | N/A | N/A | 11%-90.5% |
|---|---|---|---|---|---|---|---|
| Source | |||||||
| incidence rate | |||||||
| for | |||||||
| rearrangement | |||||||
| (95% CI) | 0.4% | 2% | 0.3% | 1.3% | 1.5% | 1% | |
| Expected range from | |||||||
| literature | 4% | ||||||
| (1.10% to | |||||||
| 9.93%) | |||||||
| 0.3% - 2.0% |
Additional supporting data
To further support the performance of AML1/ETO (RUNX1/RUNX1T1) and CBFB (CBFB) /MYH11 probes, additional clinical data sets covering the full range of the signal distribution were provided to demonstrate concordance between FISH and G-band. 100% agreement were observed for both data sets expect for one specimen that falls into re-test zone. Tables 33 and 34 demonstrate acceptable clinical concordance.
Table 33. Concordance between the AML1/ETO (RUNX1/RUNX1T1) Probe kit and Karyotyping
| AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual
Fusion FISH probe kit
| % agreement (n) | ||
|---|---|---|
| G-band result | Normal | Abnormal |
| No t(8;21) rearrangement | (b)(4) | |
| Confirmed t(8;21) rearrangement |
Table 34. Concordance between the CBFβ (CBFB) /MYH11 Probe kit and Karyotyping
| | CBFβ (CBFB) /MYH11 Translocation, Dual Fusion FISH
Probe Kit % agreement (n) | |
|---------------------------------------------|---------------------------------------------------------------------------------|----------|
| G-band result | Normal | Abnormal |
| No inv(16)/t(16;16)
rearrangement | (b)(4) | |
| Confirmed inv(16)/t(16;16)
rearrangement | | |
- The discordance was determined to be a sample with results very near the cut-off.
References:
-
- Bacher U, Kern W, Schnittger S, Hiddemann W, Schoch C, Haferlach T. Further correlations of morphology according to FAB and WHO classification to cytogenetics in de novo acute myeloid leukemia: A study on 2,235 patients. Ann Hematol. 2005;84(12):785–91.
29
-
- Bernasconi P, Klersy C, Boni M, Cavigliano PM, Calatroni S, Giardini I, et al. Incidence and prognostic significance of karyotype abnormalities in de novo primary myelodysplastic syndromes: a study on 331 patients from a single institution. Leukemia. 2005:19(8):1424-31.
-
- Byrd JC. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002 Dec 15:100(13):4325-36.
-
- Dores, G.M. et al., 2011. Acute leukemia incidence and patient survival among children and adults in the United States, 2001-2007. Blood, 119(1), pp.34-43.
-
- Grimwade, D. et al., 2010. Refinement of cytogenetic classification in acute myeloid leukemia: Determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood, 116(3), pp.354-365.
-
- Haase D. Cytogenetic features in myelodysplastic syndromes. Ann Hematol. 2008 Jul:87(7):515-26.
-
- Lazarevic V, Hörstedt A-S, Johansson B, Antunovic P, Billström R, Derolf Å, et al. Incidence and prognostic significance of karyotypic subgroups in older patients with acute myeloid leukemia: the Swedish population-based experience. Blood Cancer J. 2014 Feb 28:4(2):e188.
-
- Papaemmanuil, E. et al., 2016. Genomic Classification and Prognosis in Acute Myeloid Leukemia. New England Journal of Medicine, 374(23), pp.2209-2221.
-
- Pozdnyakova O. Miron PM. Tang G. Walter O. Raza A. Woda B. et al. Cytogenetic abnormalities in a series of 1,029 patients with primary myelodysplastic syndromes: a report from the US with a focus on some undefined single chromosomal abnormalities. Cancer. 2008 Dec 15:113(12):3331-40.
-
- Sanderson RN, Johnson PRE, Moorman a V, Roman E, Willett E, Taylor PR, et al. Population-based demographic study of karyotypes in 1709 patients with adult acute myeloid leukemia. Leukemia. 2006 Mar 19:20(3):444-50.
-
- Schanz, J. et al., 2012. New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia after MDS derived from an international database merge. Journal of Clinical Oncology, 30(8), pp.820-829.
-
- Wang XQ, Ryder J, Gross SA, Lin G, Irons RD. Prospective analysis of clinical and cytogenetic features of 435 cases of MDS diagnosed using the WHO (2001) classification: a prognostic scoring system for predicting survival in RCMD. Int J Hematol. 2009 Oct:90(3):361-9.
-
- Zhao X, Li S, Li N, Fan R, Lin G, Wang X. 11q23 abnormalities in adult Chinese patients with hematological malignancies. Med Oncol. 2014 Aug;31(8):115
-
- Clinical cut-off
Not applicable
- న. Expected values/Reference range: Same as Assay Cut-off (Upper Reference Limit) (section 1.f) above
30
N. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:
The labeling supports the decision to grant the De Novo request for this device
O. Patient Perspective
This submission did not include specific information on patient perspectives for this device.
| Identified Risks to Health | Mitigation Measures |
|---|---|
| Incorrect test results | Special controls (1), (2), (3), and (4) |
| Incorrect interpretation of test results | Special controls (1), (2), (3), and (4) |
P. Identified Risks to Health and Mitigation Measures:
O. Benefit/Risk Summary:
Clinical reviewer memo will contain details for benefit risk determination.
| Summary of the
Benefits | Summary of the
Risks | Summary of Other
Factors | Conclusions |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| AML and MDS
patients may benefit by
use of the device on
bone marrow
specimens to obtain
results that can be used
in accordance with the
World Health
Organization criteria
for assessment of these
patient specimens
when assay results are
interpreted by a
qualified pathologist or
cytogeneticist. | Erroneous device
performance can yield
false negative or false
positive results or
incorrect interpretation
of test results by the
user which may
adversely influence
management of AML
or MDS patients. | Risks are mitigated by
analytical and clinical
validation studies using
the device probes, along
with labeling and
supports the intended
use. The device use
requires a qualified
pathologist or
cytogeneticist in the
context of
histopathological
evaluation (e.g.,
immunohistochemistry). | Yes. Based on the
supporting clinical
studies for the
diagnostic device
along with review of
the analytical
performance and
labeling, the probable
benefits outweigh the
probable risks in light
of the mitigations
provided by the special
controls, in addition to
the general controls. |
S. Conclusion:
The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 864.1880. FDA believes that the stated special controls, in combination with the general controls, provide a reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:
Product Code: QDI
31
| Device Type: | Fluorescence in-situ hybridization based detection of chromosomal
abnormalities from patients with hematologic malignancies |
|--------------|--------------------------------------------------------------------------------------------------------------------------------|
| Class: | II (Special Controls) |
| Regulation: | 21 CFR 864.1880 |
- (a) IDENTIFICATION: Fluorescence in-situ hybridization based detection of chromosomal abnormalities from patients with hematologic malignancies is used to detect chromosomal abnormalities in human specimens from patients with hematologic malignancies. The test is indicated for the clinical management of patients consistent with internationally accepted guidelines (e.g. World Health Organization guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues) and in conjunction with other clinical and clinicopathological criteria. The results are to be interpreted by a pathologist or equivalent professional.
- (b) CLASSIFICATION: Class II (special controls). The special controls for this device are:
- (1) Design verification and validation (b)(4) -must include:
- (i) A detailed description of all probes included in the kit;
- Purpose of each probe; (ii)
- (iii) Probe molecular specificity;
- (iv) Probe specificity:
- Probe limits: (v)
- Probe sensitivity; (vi)
- Specification of required ancillary reagents, instrumentation, and equipment; (vii)
- Specification of the specimen collection, processing, storage and slide (viii) preparation methods;
- Specification of the assay procedure: (ix)
- Specification of control elements that are incorporated into the recommended (x) testing procedures;
- Specification of the criteria for test result interpretation and reporting; (xi)
- (xii) Documentation demonstrating analytical validation that includes:
- (A)Device analytical sensitivity data with a minimum of 25 specimens from karyotypically normal males.
- (B)Device analytical specificity data with a minimum of 5 specimens from karyotypically normal males.
- (1) Design verification and validation (b)(4) -must include:
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- (C) Description of how the clinical threshold was assigned and verification of the assigned clinical threshold.
- (D) Device precision/reproducibility data with a minimum of 6 clinical specimens including 2 negative specimens, 2 positive specimens near the clinical decision threshold (cut-off) and 2 positive specimens. The data must include results obtained from 3 sites (as applicable), with 2 operators at each site, with the assay run for a minimum of 3-5 non-consecutive days and each specimen run in duplicate for a minimum of 30 replicates.
- (E) Between-reagent lot reproducibility using 3 reagent lots and 3 clinical specimens representing negative, near cut-off /low positive, and positive.
- (F) Device stability data to include:
- (1) Real-time Stability,
- (2) Freeze-Thaw Stability,
- (3) Transport and Temperature Stability, as applicable,
- (4) Post-Hybridization Signal Stability, and
- (5) Photostability of Probe.
- (xiii) Documentation demonstrating the clinical validity of the device that includes:
- (A) A summary of the prevalence and clinical thresholds reported in 3 peerreviewed published literature references for the intended use population of the device and device performance data demonstrating conformance with the published prevalence as reported in peer-reviewed published literature references based on testing clinical specimens, selected without bias (e.g., consecutively selected) from the intended use population using the specific device seeking marketing clearance. A minimum number of clinical specimens must be tested to ensure sufficient positives are evaluated by the device, or alternatively, in the absence of a sufficient number of positives, an additional comparison of results obtained with the device to clinical truth (e.g., confirmed clinical diagnosis and/or G-banded karyotyping) with an independent specimen set must be conducted.
- (B) Documentation for peer-reviewed published literature references must include the following elements:
- (1) Whether the specific device was used in the literature reference;
- (2) Number and type of specimens;
- (3) Target population studied;
- (4) Upper reference limit; and
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- (5) Prevalence range estimated based on the number of positive probe results.
- (C) In the absence of clinical data obtained from paragraphs (b)(1)(xiii)(A) and (b)(1)(xiii)(B) of this section, clinical data obtained from a method comparison to the predicate with positives and negative clinical specimens.
- (2) The intended use required on the label under 21 CFR 809.10(a)(4) and on the labeling under 21 CFR 809.10(b)(5)(ii), must include a statement that
"The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic."
- (3) The 21 CFR 809.10(b) labeling must include information that demonstrates the performance characteristics of the test, including a detailed summary of the performance studies conducted and their results, as described in paragraphs (b)(1)(iv) through (b)(1)(xiii) of this section. The 21 CFR 809.10(b) labeling must include the pre-specified acceptance criteria for these performance studies, justification for the pre-specified acceptance criteria, and whether the prespecified acceptance criteria were met.
- (4) The 21 CFR 809.10(b) labeling must include the following limiting statements:
- "Reporting and interpretation of FISH results should be consistent with (i) professional standards of practice and should take into consideration other clinical and diagnostic information. This kit is intended as an adjunct to other diagnostic laboratory tests and therapeutic action should not be initiated on the basis of the FISH result alone. Failure to adhere to the protocol may affect the performance and lead to false results."
- (ii) "Each lab is responsible for establishing their own cut-off values. Each laboratory should test sufficiently large number of samples to establish normal population distribution of the signal levels and to assign a cut-off value. The product is for professional use only and is intended to be interpreted by a qualified Pathologist or Cytogeneticist."