Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN. Key Words: .... ITable 1I. List of AML FISH Probes and Targets. Targeted Abnormality.
AJCP / Original Article
Conventional Karyotyping and Fluorescence In Situ Hybridization An Effective Utilization Strategy in Diagnostic Adult Acute Myeloid Leukemia Rong He, MD,1 Anne E. Wiktor,2 Curtis A. Hanson, MD,1 Rhett P. Ketterling, MD,2 Paul J. Kurtin, MD,1 Daniel L. Van Dyke, PhD,2 Mark R. Litzow, MD,3 Matthew H. Howard, MD,1 and Kaaren K. Reichard, MD1 From the Divisions of 1Hematopathology and 2Laboratory Genetics, Department of Laboratory Medicine and Pathology, and 3Division of Hematology, Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN. Key Words: Hematopathology; Hematology; Genetics; Molecular diagnostics Am J Clin Pathol June 2015;143:873-878 DOI: 10.1309/AJCPP6LVMQG4LNCK
ABSTRACT Objectives: Cytogenetics defines disease entities and predicts prognosis in acute myeloid leukemia (AML). Conventional karyotyping provides a comprehensive view of the genome, while fluorescence in situ hybridization (FISH) detects targeted abnormalities. The aim of this study was to compare the utility of karyotyping and FISH in adult AML. Methods: We studied 250 adult AML cases with concurrent karyotyping and FISH testing. Karyotyping was considered adequate when 20 or more metaphases were analyzed. Results: In total, 220 cases had adequate karyotyping and were classified as normal karyotype/normal FISH (n = 92), normal karyotype/abnormal FISH (n = 4), abnormal karyotype/normal FISH (n = 8), and abnormal karyotype/ abnormal FISH (n = 116). The overall karyotype/FISH concordance rate was 97.7% with five discordant cases identified, four from the normal karyotype/abnormal FISH group and one from the abnormal karyotype/abnormal FISH group. No karyotype/FISH discordance was seen in the abnormal karyotype/normal FISH group for the FISH probes evaluated. FISH lent prognostic information in one (0.5%) of 220 cases with normal karyotype/abnormal FISH: CBFB-MYH11 fusion, indicating favorable prognosis. Conclusions: In adult AML, FISH rarely provides additional information when karyotyping is adequate. We therefore propose an evidence-based, cost-effective algorithmic approach for routine conventional karyotype and FISH testing in adult AML workup.
© American Society for Clinical Pathology
Acute myeloid leukemia (AML) is an aggressive myeloid neoplasm that, without clinical intervention, generally pursues an acutely progressive clinical course. Given the morphologic, immunophenotypic, and genetic heterogeneity of AML, the World Health Organization (WHO) has recently classified AML into four broad categories: AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related myeloid neoplasms, and AML, not otherwise specified.1 Conventional karyotyping is the cornerstone of risk stratification in AML, resulting in favorable, unfavorable, and intermediate-risk prognostic groups.2 Patients in the favorable risk group include those with AML-defining t(15;17)(q24.1;q21), PML-RARA; t(8;21)(q22;q22), RUNX1RUNX1T1; and inv(16)/t(16;16)(p13.1;q22) or t(16;16) (p13.1;q22), CBFB-MYH1 and usually respond well to chemotherapy-based consolidation. In contrast, patients with unfavorable cytogenetics often require allogeneic stem cell transplant to improve outcome and survival. Intermediaterisk cytogenetic findings are encountered in approximately 50% of AML cases, with the majority having a normal karyotype. An abundance of recent molecular studies have identified multiple novel genetic abnormalities such as FLT3ITD, NPM1, and CEBPA mutations that further delineate the molecular pathogenesis and refine risk stratification in AML, particularly in the normal karyotype group.3-7 Currently, recommended genetic testing at the time of AML diagnosis includes conventional karyotype analysis and molecular genetic testing for FLT3, NPM1, and CEBPA mutations, with the latter being particularly important when
Am J Clin Pathol 2015;143:873-878 873 DOI: 10.1309/AJCPP6LVMQG4LNCK
He et al / Conventional Karyotyping and Fluorescence In Situ Hybridization
❚Table 1❚ List of AML FISH Probes and Targets Targeted Abnormality
FISH Probe
t(8;21)(q22;q22) t(15;17)(q24.1;q21) inv(16)/t(16;16)(p13.1;q22) t(6;9)(p23;q34) t(9;22)(q34;q11.2) inv(3)(q21.3;q26.2) 11q23 rearrangement +8 13q– 5q–/–5 7q–/–7 20q–
RUNX1-RUNX1T1 PML-RARA CBFB-MYH11 DEK-NUP214 BCR-ABL1 RPN1-MECOM MLL D8Z2-MYC D13S319-LAMP1 EGR1-D5S630 D7Z1-D7S486 D20S108/20qter
AML, acute myeloid leukemia; FISH, fluorescence in situ hybridization.
a normal karyotype is identified.1,8 It is also common clinical practice to supplement the aforementioned genetic tests with fluorescence in situ hybridization (FISH) studies that target specific recurring genetic abnormalities. With the continuing growth of knowledge regarding molecular mutations in AML and their associated prognostic and therapeutic implications, the list of recommended genetic tests in AML will certainly evolve and change over time. The current health care system emphasizes efficiency and value. Since genetic testing in AML is essential but often expensive, the challenge for pathologists, hematologists, and geneticists is to ensure that only appropriate testing is performed and that it is informative, cost-effective, and clinically relevant without being redundant. Thus, the purpose of the study was to determine the value of performing FISH studies in adult patients with AML when done in conjunction with karyotyping. Ultimately, the unique aim of our study was to devise an evidence-based, cost-effective algorithmic approach for conventional karyotype and FISH testing in adult AML.
Materials and Methods We identified adult Mayo Clinic patients evaluated for possible AML between September 2006 and July 2013. All patients had a bone marrow aspiration and biopsy performed. We evaluated each patient’s clinical history and hematopathology report. The diagnosis was based on morphologic review of the bone marrow and/or flow cytometric immunophenotyping, as well as WHO criteria for AML.1 Patients who fulfilled the WHO diagnostic criteria of AML were included; those who did not were excluded from further analysis. Karyotype and FISH testing were performed on each bone marrow sample by the Mayo Clinic Cytogenetics
874 Am J Clin Pathol 2015;143:873-878 DOI: 10.1309/AJCPP6LVMQG4LNCK
Laboratory. FISH analysis included probes for RUNX1RUNX1T1, PML-RARA, MLL, CBFB-MYH11, and, in some cases, DEK-NUP214, BCR-ABL1, RPN1-MECOM, +8, 13q–, 5q–/–5, 7q–/–7, or 20q– ❚Table 1❚. Fresh bone marrow aspirate samples were cultured and harvested following standard cytogenetic methods, and chromosome preparations were stained using GTL banding with trypsin and Leishman stain. FISH analysis was performed following laboratory-validated protocols using commercial or laboratory-developed probes. A total of 500 nuclei were scored for dual-color, dual-fusion (D-FISH) probes designed to identify translocations or inversions (eg, PML-RARA, CBFB-MYH11) and 200 nuclei for probes detecting copy number changes (eg, +8) or break-apart probes detecting gene disruption (eg, MLL). The analysis was split between two technologists for each probe set. For each bone marrow specimen, we recorded the karyotype indicating the number of normal and abnormal metaphases analyzed and the percentage of abnormal interphase nuclei identified with each FISH probe (when it exceeded the normal cutoff). The chromosome results were classified as normal, nonclonal, or abnormal based on the International System for Human Cytogenetic Nomenclature (2013) criteria.9 A specimen was considered normal for any of the following results: no abnormality detected, isolated loss of a sex chromosome, trisomy 15 with or without loss of a sex chromosome, or a constitutional rearrangement as a sole abnormality.10,11 An abnormality was considered nonclonal when less than two metaphases with a chromosome gain or structural rearrangement or less than three cells with a chromosome loss were identified. Cases with a nonclonal karyotype result were included in the normal karyotype category. The karyotype study was considered adequate when there were 20 or more metaphases available for review. Karyotyping was considered insufficient when only one to 19 metaphases were identified. This project was approved by the Mayo Clinic Institutional Review Board.
Results A total of 250 adult patients meeting the WHO diagnostic criteria of AML were identified who had a bone marrow evaluation and concurrent karyotype and AML FISH studies. Karyotyping was successfully performed on 244 (98%) of 250 cases, and the other six of 250 cases had no metaphase growth for chromosome analysis. Of the 244 cases, 220 (90%) were adequate for karyotype studies (≥20 metaphases), while 12 cases (
