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Application Note - Reducing your FISH probe validation burden

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Introduction

This application note describes the detailed clinical and analytical studies carried out to meet the performance required to achieve FDA clearance of the eight probes for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) listed in Table 1. In addition, we present data demonstrating excellent analytical reproducibility of the probes and extensive stability studies. During the course of these studies, which spanned four sites, worldwide, over 2,500 replicates were run with no technical failures. The rigorous standards required for gaining FDA clearance are recognized globally and emphasize the exceptional quality of the Cytocell FISH probes from Oxford Gene Technology (OGT).

Table 1. The 8 FISH probes in this studyTable 1. The eight FISH probes in this study

The Cytocell Aquarius AML/MDS range of FISH probe test kits are fluorescence in situ hybridization (FISH) tests used to detect common chromosomal rearrangements in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The tests are indicated for the characterization of patient specimens consistent with World Health Organization guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are to be interpreted by a qualified pathologist or cytogeneticist. The tests are not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

Refer to individual test kit Package Insert for the specific intended use and limitations.

For In Vitro Diagnostic Use. Rx only.

Clinical background

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, whilst MDS is characterized by the simultaneous proliferation and apoptosis of hemopoietic cells1. According to the World Health Organization (WHO), the global incidence for MDS is 3-5 cases per 100,000 (non-age corrected) with approximately 10,000 new cases of MDS diagnosed annually in the USA1. The Surveillance Epidemiology and End Results (SEER) statistics present a similar picture for AML with a USA incidence of 4.3 per 100,000 (non-sex specific)2.

The National Comprehensive Cancer Network Guidelines in Oncology (NCCN Guidelines®) recognizes that FISH testing is a valuable tool in determining and clarifying the presence of common chromosomal rearrangements in the investigation of AML and MDS. The information from cytogenetic analysis is used for clinical management of patients consistent with WHO guidelines in AML3 and the prognostic scoring systems of MDS4. FISH testing can also be used to monitor patient progress during and after treatment for AML and MDS, by assessing the presence or absence of an abnormality within a bone marrow sample5,6.

Study Overview and Results

An extensive range of studies were conducted to determine the performance characteristics of the Cytocell Aquarius AML/MDS FISH Probe Kits. All Cytocell FISH probes are designed to give tight, bright signals that are easy-to-score with minimal background, enabling rapid analysis and reduced repeat testing.

An example of probe design and typical results is shown in Figure 1.

Figure 1. Example of the results obtained for USA LPH 036 EVI1 (MECOM) v2Figure 1. Example of the results obtained for USA-LPH 036: EVI1 (MECOM), including [A] the probe design, [B] expected normal 2RGB, [C] a sample showing an inversion involving the MECOM region 1B/1RG/1RGB and [D] a sample showing a translocation involving the MECOM region 1R/1GB/1RGB.

Analytical Specificity

Analytical specificity can be defined as the percentage of signals that hybridize to the correct locus in a metaphase spread and at no other location7. In our study, all eight probes were shown to have an analytical specificity of 100% (with upper and lower 95% confidence levels of 100% and 98.12% respectively) in a total of 1600 loci that were examined across all eight probe sets8. The high level of analytical specificity seen demonstrates that these probes will allow confident probe signal interpretation with no cross-hybridization.

Analytical Sensitivity

Analytical sensitivity can be described as the percentage of chromosome targets or interphase nuclei with the expected normal signal pattern in a negative cell sample for that abnormality. This is a measure of how well the probe detects the target sequence7. In this study, the FISH probes were hybridized to normal specimens and the number of expected normal signal patterns seen was expressed as a percentage.

A FISH probe exhibiting good hybridization would be expected to have an analytical sensitivity of at least 95%. All eight FISH probes evaluated in this study showed an analytical sensitivity of greater than 98% indicating that these FISH probes provide reliable, consistent results, enabling fast accurate analysis to be performed.

Table 2. Analytical sensitivity of the eight FISH probes in this studyTable 2. Analytical sensitivity of the eight FISH probes in this study

Clinical studies

The clinical studies compared the incidence rates of each of the positive rearrangements indicated by the eight probes being evaluated, with the incidence rates reported in published literature (Table 3). All the FISH probes evaluated demonstrated the expected incidence when compared to reported values in the literature (within 95% confidence limits).

The USA-LPH 026 AML/ETO study results did not reveal any rearrangements of the target chromosome within one population sample and required an additional clinical evaluation. To assess the clinical performance of the probe, ten known samples containing the rearrangement were blinded into a cohort of 90 additional samples known to be negative for the rearrangement. All samples were identified correctly. The USA-LPH 026 AML/ETO probe met the acceptance criteria of this supplementary study.

Table 3. Summary of clinical study resultsTable 3. Summary of clinical study results. Confirmation that the Cytocell AML/MDS FDA cleared probes detected positive genetic aberrations in concordance with expected genetic aberration prevalence rates from the literature.

Reproducibility

Reproducibility is a measurement of the consistency of the FISH test when performed at different times by different operators. A highly reproducible test is important to ensure confidence in achieving reliable FISH results with minimal variability in probe performance.

In our study we considered several different types of reproducibility, sample-to-sample (same day), site-to-site, day-to-day and lot-to-lot variability. All eight probes met or exceeded the acceptance criteria of >95% agreement across all variables for intra/inter-day and inter-site reproducibility, demonstrating exceptional consistency (Table 4). Further testing was carried out to demonstrate inter-lot reproducibility.

Table 4. Summary of the reproducibility results achievedTable 4. Summary of the reproducibility results achieved.

The intra-day reproducibility covered sample-to-sample variability. Inter-day considered variability between separate days. Inter-site reproducibility was measured across three testing sites. Inter-lot measured probe batch-to-batch variability. These variables were jointly analyzed to show an overall reproducibility of these probes. A negative class cell sample is defined as negative for the supported signal pattern. A high positive class cell sample type represents samples that show >45% cell positivity for the supported signal pattern.

Establishing cut-off value

The cut-off value (upper reference limit) can be considered as the percentage of scoreable interphase cells with a specific abnormal FISH signal pattern found in a normal cell sample for that abnormality. Good validation practice suggests that cut-offs should be generated from a pool of at least 20 – 25 karyotypically normal bone marrow samples or samples that are negative for the abnormality which the probe is detecting7,22,23,24.

For each probe, varying numbers of samples were used to calculate the normal cut-off values (Table 5). The cutoffsfor all probes were calculated using the BETAINV method, a widely used statistical method for the determination of the normal cut-off 23,24.

Table 5. Summary of the Normal Cut Off (or Upper Reference Limit)Table 5. Summary of the Normal Cut-Off (or Upper Reference Limit)

Probe stability

It is important that any tests used in a diagnostic setting maintain their performance under standard laboratory conditions. Extensive stability studies with a range of temperatures and treatments were performed to assess the FISH probe stability.

It is also sometimes necessary to review slides once the initial analysis has been completed, therefore the assessment of the integrity of final analyzed FISH slides was studied. The stability studies performed are described below and all eight probes maintained their performance.

24-month shelf life

The FISH probes were stored at -20°C in the dark, an isochronal study was performed where probe lots of different ages were assessed on identical samples. In order to support a 24-month shelf life, the minimum age of lots to be assessed was 25 months; however, lots of probes between 0-34 months were included to provide supplementary data in support of the statistical analysis. All probes met the requirements for a 24-month shelf life claim.

Freeze/thaw stability

Probes were subjected to 11 rounds of thawing and freezing, subsequent FISH testing showed the probes to be stable after a total of 11 freeze/thaw cycles.

Transportation stability

A transport study was conducted at temperatures of 40°C for two weeks, subsequent FISH testing demonstrated stability for all probes.

Hybridized slide stability

When stored in darkness at 2-8°C for up to one month, hybridized slides demonstrated reproducible analyzability.

Light exposure stability

All probes were shown to be stable under limited laboratory light exposure as might be expected during the normal use of FISH probes.

Conclusion

In summary, in order to fulfill the high standards required for an FDA submission, all eight Cytocell FISH probes in this submission underwent stringent performance assessments to underpin the probe performance and ensure that the products were safe and effective for their intended use.

FDA clearance provides you with the confidence that, when choosing FDA-cleared Cytocell probes from OGT, the following benefits are delivered to your laboratory:

1. Reduction in validation burden

Following the extensive analytical and clinical studies, all probes met the stringent acceptance criteria required to demonstrate probe specificity, sensitivity and clinical utility. This provides confidence that these probes can meet their clinical intended use and reduces the validation burden in your laboratory.

2. Decreased FISH retest rates

The performance of the FDA-cleared AML/MDS probes has been demonstrated to be highly reproducible, delivering high intensity signals with excellent contrast. Consequently, the performance of these probes enables reliable, accurate analysis and low retest rates which can help streamline operations in your laboratory.

3. Minimized hands-on-time and reduction in handling errors

Cytocell FDA-cleared probe kits are shipped in an easy-to-use, pre-mixed probe format and are supplied with DAPI, along with detailed protocols and analysis guidelines. The kits help to simplify processing and minimize the chance for error, adding further efficiencies to the FISH workflow.

4. Access to expert, technical support when you need it

Our quality products are all backed by expert support from our technical specialists enabling you to focus on delivering high quality, rapid test results to the patient.

For additional details regarding the experimental design and criteria used, please refer to the relevant Package Insert (Instructions For Use, IFU) document provided with the product.

References

1. Swerdlow et al., editors, WHO Classifcation of Tumors of Haematopoietic and Lymphoid Tissue, Lyon, France, 4th edition, IARC,2017.

2. Noone AM, Howlader N, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2015, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2015/, based on November 2017 SEER data submission, posted to the SEER web site, April 2018.

3. National Comprehensive Cancer Network. Acute Myeloid Leukemia (Version 3.2017) [Internet]. [cited 2017 Sep 9]. Available from:https://www.nccn.org/professionals/physician_gls/f_guidelines.asp#aml.

4. National Comprehensive Cancer Network. Myelodysplastic Syndromes (Version 1.2018) [Internet]. [cited 2017 Sep 9]. Available from:https://www.
nccn.org/professionals/physician_gls/f_guidelines.asp#mds.

5. Pavletic SZ, Kumar S, Mohty M, de Lima M, Foran JM, Pasquini M, et al. NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on the Epidemiology and Natural History of Relapse following Allogeneic Cell Transplantation. Biol Blood Marrow Transplant. 2010;16(7):871–90.

6. Shah NN, Bacher U, Fry T, Calvo KR, Stetler-Stevenson M, Arthur DC, et al. Myelodysplastic syndrome after allogeneic hematopoietic stem cell transplantation: Diagnostic and therapeutic challenges. Am J Hematol. 2012;87(9):916–22.

7. Wiktor AE, Van Dyke DL, Stupca PJ, et al. Preclinical validation of fluorescence in situ hybridization assays for clinical practice. Genet Med. 2006;8(1):16-23.

8. Wilson, EB. Probable inference, the law of succession, and statistical inference. J. Am. Stat. Assoc.1927;22:209–212.

9. Papaemmanuil, E. et al., 2016. Genomic Classification and Prognosis in Acute Myeloid Leukemia. New England Journal of Medicine, 374(23), pp.2209–2221.

10. 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.

11. 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.

12. 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.

13. 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.

14. Haase D. Cytogenetic features in myelodysplastic syndromes. Ann Hematol. 2008 Jul;87(7):515–26.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. Mascarello JT, Hirsch B, Kearney HM, et al. Section E9 of the American College of Medical Genetics technical standards and guidelines: fluorescence in situ hybridization. Genet Med. 2011;13(7):667-675.

23. Arsham, MS., Barch, MJ. and Lawce HJ. (eds.) (2017) The AGT Cytogenetics Laboratory Manual. New Jersey: John Wiley & Sons Inc.

24. Wolff DJ, Bagg A, Cooley LD et al. Guidance for Fluorescence in Situ Hybridisation Testing in Hematologic Disorders. J Med Diagnost. 2007;9(2):134-143.

This document and its contents are © Oxford Gene Technology IP Limited – 2019. All rights reserved. Trademarks: OGT™ (Oxford Gene Technology IP Ltd); Cytocell®, Aquarius® (Cytocell Ltd), NCCN® Guidelines (National Comprehensive Cancer Network Inc.) Cytocell FDA cleared probes: For In Vitro Diagnostic Use. Rx only. Product availability may vary from country to country and is subject to varying regulatory requirements. Please contact your local representative for availability