A Comparative Analysis of Molecular Genetic and Conventional Cytogenetic Detection of Diagnostically Important Translocations in More than 400 Cases of Acute Leukemia, Highlighting the Frequency of False-negative Conventional Cytogenetics

Rebecca L. King, MD; Mojdeh Naghashpour, MD, PhD; Christopher D. Watt, MD, PhD; Jennifer J.D. Morrissette, PhD; Adam Bagg, MD


Am J Clin Pathol. 2011;135(6):921-928. 

In This Article

Abstract and Introduction


In this study, we correlated the results of concurrent molecular and cytogenetic detection of entity-defining translocations in adults with acute leukemia to determine the frequency of cryptic translocations missed by conventional cytogenetics (CC) and of recurrent, prognostically relevant translocations not detectable by multiplex reverse transcriptase–polymerase chain reaction (MRP). During a 5.5-year period, 442 diagnostic acute leukemia specimens were submitted for MRP-based detection of 7 common recurrent translocations: t(8;21), t(15;17), inv(16), t(9;22), t(12;21), t(4;11), and t(1;19), with a detection rate of 15.2% (67/442). CC was performed in 330 (74.7%) of 442 cases. In 7 of these 330 cases, CC missed the translocation detected by MRP. In 50 additional cases, CC revealed 1 of the MRP-detectable translocations (all were also MRP positive), yielding a false-negative rate of 12% (7/57) for the CC assay. The remaining 140 of 190 cases with clonal cytogenetic changes harbored abnormalities that were not targeted by the MRP assay, including 8 that define specific acute myeloid leukemia entities. This study revealed the frequent occurrence of false-negative, entity-defining CC analysis and highlighted the complementary nature of MRP and CC approaches in detecting genetic abnormalities in acute leukemia.


The contemporary diagnosis of acute leukemia (AL) relies on a multifaceted approach using morphologic, cytochemical, immunophenotypic, and cytogenetic and molecular analysis.[1] Although each of these modalities provides critical information with regard to diagnosis, it has been well established in recent years that genetic studies provide the most disease-defining, prognostically relevant, and therapy-determining set of data.[1–14] ALs harbor a variety of recurrent genetic aberrations, some of which can be ascertained by conventional cytogenetic (CC) analysis.[2–7,14–16] These comprise 3 broad categories of abnormalities: (1) balanced chromosomal translocations, typically, but not always, without a net gain or loss of genetic material; (2) numeric abnormalities, such as deletions and additions of whole or segments of chromosomes; and (3) submicroscopic genetic abnormalities. A subset of balanced translocations in ALs are diagnostic of specific entities and have independent prognostic value, which directly impacts therapeutic decision making.[1,3,4]

Although many of the translocations can be detected (and indeed were discovered) by CC, newer molecular and fluorescence in situ hybridization (FISH) methods for detection of specific abnormalities are becoming routine in the clinical laboratory.[17] Molecular methods, specifically reverse transcriptase–polymerase chain reaction (RT-PCR)–based assays, have numerous technical advantages over CC, including shorter turnaround time and no requirement for dividing cells. In addition, prognostically significant translocations may go undetected by CC if they involve regions with similar banding patterns, so-called cryptic translocations.[16,18–24] Thus, RT-PCR and FISH also have the benefit of detecting aberrations that may be missed by CC analysis.

Studies have demonstrated the excellent diagnostic sensitivity of FISH for detecting translocations and numeric aberrations in AL and have encouraged its use as an adjunct to CC.[25,26] FISH, as well as RT-PCR, can be multiplexed, allowing for the investigation of several chromosomal aberrations at once.[25–28] Multiplex FISH also provides the unique benefit of clarifying complex karyotypes with identification of derivative chromosomes, ring chromosomes, and complex translocations involving more than 2 chromosomes that may not be apparent by CC.[25–28] However, multiplex FISH is not currently used in routine diagnostic laboratories.

RT-PCR also provides a molecular fingerprint that can be precisely, sensitively, and quantitatively measured, allowing for the subsequent posttherapeutic detection of minimal residual disease.[16,19,29,30] Submicroscopic abnormalities, such as point and length mutations, are assuming increased relevance in AL[31] and, by definition, can be detected only by molecular approaches. However, despite these obvious advantages, molecular approaches, including RT-PCR–based assays, have not obviated the role for CC (or FISH) studies in the diagnosis and characterization of AL. In addition to detecting the common, relevant translocations, CC has the advantage of identifying numeric aberrations and rare translocations that are not yet well characterized or readily amenable to molecular detection. Indeed, some of these lesions, such as t(1;22), t(6;9), and inv(3), have been incorporated in the World Health Organization (WHO) 2008 classification of hematopoietic malignancies.[1]

The validation and initial application to routine molecular diagnostic practice of the detection of common prognostically relevant leukemia-associated translocations using multiplex RT-PCR (MRP) have been reported.[32,33] The goal of the present study was to detail our experience in a larger cohort of patients during an extended period to evaluate the relative roles of MRP and CC in the classification of AL. In doing so, we sought to establish the usefulness of each method for detecting diagnostically and prognostically relevant translocations. Finally, we wanted to determine the frequency of cryptic translocations missed by CC and of recurrent, prognostically relevant translocations not detectable by the MRP assay.


Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.
Post as: