Comparison of Diagnostic Yield Among M-FISH, FISH Probe Panel and Conventional Cytogenetic Analysis in AML

Overview

Conventional cytogenetic studies have been the gold standard for more than five decades for detecting genetic alterations that are greater than 10 Mb (mega base pairs) in size. Conventional cytogenetic studies have paved the way in identifying specific chromosomal aberrations associated with clinically and morphologically definitive subsets of hematological neoplasms. Fluorescence in situ hybridization (FISH) has become a reliable and rapid complementary test in targeting critical genetic events associated with diagnostics and prognosis in hematological neoplasms. In the current health care environment, which increasingly focuses on value and efficiency, it is critical for pathologists and clinicians to effectively navigate this environment and judiciously incorporate these high-complexity and expensive techniques into routine patient care. While conventional karyotyping provides a comprehensive view of the genome, FISH can detect cryptic or submicroscopic genetic abnormalities and identify recurrent genetic abnormalities in nondividing cells. As a consequence, it is commonly extrapolated that FISH will improve the sensitivity of detecting all genetic abnormalities compared with conventional karyotyping analysis. This assumption has then been translated in clinical practice to having clinicians and pathologists routinely ordering both conventional karyotyping and FISH studies in patients with hematological neoplasms. Depending on how comprehensive the FISH panel is, the cost for this testing may be quite expensive, and its additive value remains questionable. It is common practice for laboratories to use FISH panels in conjunction with karyotyping both in diagnostic specimens and during follow-up to monitor response to therapy. Multiplex FISH (M-FISH) represents one of the most significant developments in molecular cytogenetics of the past decade. In tumor and leukemia cytogenetics, two groups have been targeted by M-FISH to identify cryptic chromosome rearrangements not detectable by conventional cytogenetic studies: those with an apparently normal karyotype (suspected of harboring small rearrangements not detectable by conventional cytogenetics) and those with a complex aberrant karyotype (which are difficult to karyotype accurately due to the sheer number of aberrations).

Neoplastic hematology is at the forefront of personal¬ized medicine. The World Health Organization (WHO) classification incorporates genetic data with microscopic, immunophenotypic, and clini¬cal findings and categorizes hematologic neoplasms into clinically and biologically distinct categories. This system guides diagnosis, prognosis and therapeutic choices, which has become increasingly important with emerging targeted therapies for lymphoma and leukemia. However, laboratory tests for hematologic neoplasms are of high complexity and are generally quite costly. The initial assessment of many hematologic neoplasms includes morphological, histological, immunophenotypic (flow cytometric and/or immunohistochemical), and conventional cytogenetic studies. Data obtained from cytogenetic analysis can be pathognomonic for specific leukemias in the WHO classification (for example, acute myeloid leukemia (AML) with recurrent cytogenetic abnormalities and chronic myelogenous leukemia (CML)) and are likely to assume a greater role in defining specific categories in further classifications. Conventional cytogenetic studies have been the gold standard for more than five decades for detecting genetic alterations that are greater than 10 Mb (mega base pairs) in size. They have paved the way in identifying specific chromosomal aberrations associated with clinically and morphologically definitive subsets of hematological neoplasms. Fluorescent in situ hybridization (FISH) has become a reliable and rapid complementary test in targeting critical genetic events associated with diagnostics and prognosis in hematological neoplasms. FISH has addressed the issues with conventional cytogenetic studies by targeting interphase cells in addition to metaphases. It has become clear that FISH studies may also be an integral component of the diagnostic evaluation, particularly where the abnormality is "cryptic" i.e. not evident by conventional cytogenetic studies. Although complementary FISH testing increases the overall detection of aberrations, its benefit is not uniform across all types of hematological neoplasms. This is because FISH probes are restricted to the detection of only specific abnormalities and genetic alterations beyond the scope of the FISH probes would therefore be completely missed. It is common practice for laboratories to use FISH probe panels in conjunction with karyotyping both in diagnostic specimens and during follow-up to monitor response to therapy. FISH is targeted toward specific abnormalities, and results can be evaluated in an automated fashion on interphase nuclei, allowing for examination of more cells than conventional cytogenetic studies. FISH has higher analytic and, in certain circumstances, higher clinical sensitivity compared with conventional cytogenetic studies. The usage of FISH probe panels in aiding diagnosis or in monitoring follow-up samples of hematologic neoplasms is critical. The Eastern Collaborative Oncology Group (ECOG) compared conventional cytogenetic studies and FISH in AML patients and found that a probe panel to detect monosomy 5/deletion 5q, monosomy 7/deletion 7q, trisomy 8, t(8;21), t(9;22), MLL rearrangements with various partners, t(15;17), and inv(16)/t(16;16), had a concordance between 98% and 100%. On the other hand, He et al study demonstrates the limited value of FISH testing in adult AML in the setting of an adequate karyotyping study. Despite of many significant technological advances made in recent years in the area of clinical genetic testing, conventional cytogenetic studies and routine FISH studies remain important laboratory testing tools available for evaluating hematologic neoplasms. Usually these two testing methods complement each other and often FISH serves to clarify and better define cytogenetic results. Therefore, there is a very strong expectation that cytogenetic and FISH results should confirm each other in spite of cases that appear to be exceptions. When conflicting results occur by these two testing methods on the same specimen, clinical laboratories are challenged to offer explanations based on empirical data beyond simply stating that it was or was not due to laboratory error. Multiplex FISH (M-FISH) represents one of the most significant developments in molecular cytogenetics of the past decade. In tumor and leukemia cytogenetics, two groups have been targeted by M-FISH to identify cryptic chromosome rearrangements not detectable by conventional cytogenetic studies: those with an apparently normal karyotype (suspected of harboring small rearrangements not detectable by conventional cytogenetics) and those with a complex aberrant karyotype (which are difficult to karyotype accurately due to the sheer number of aberrations). Zhang et al. used spectral karyotype (SKY) to re-evaluate the karyotypes of AML cases reported as normal by G-banding. This resulted in the identification of one case of t(11;19)(q23;p13), a subtle but recognized cytogenetic abnormality, and a minor clone containing monosomy for chromosome 7 in another case. In a similar study, Mohr et al. compared SKY to conventional karyotyping in patients with AML or MDS with normal karyotypes. No abnormalities were identified. With the lack of an evidence-based standardized algorithmic approach, misuse and overutilization of laboratory tests are common and result in increased health care costs and patient care complexity. In the current health care environment, which increasingly focuses on value and efficiency, it is critical for pathologists and clinicians to incorporate high-complexity and expensive techniques into routine patient care. While conventional cytogenetic studies provide a comprehensive view of the genome, FISH probe panel can detect cryptic or submicroscopic genetic abnormalities and identify recurrent genetic abnormalities in nondividing cells. M-FISH can identify cryptic chromosome rearrangements that are not detected by conventional cytogenetic studies. As a consequence, it is commonly extrapolated that FISH will improve the sensitivity of detecting all genetic abnormalities compared with conventional cytogenetic studies. This assumption has then been translated in clinical practice to having clinicians and pathologists routinely ordering both conventional cytogenetic studies and FISH studies in patients with hematological neoplasms. Depending on how comprehensive the FISH probe panel is, the cost for this testing may be quite expensive, and its additive value remains questionable.